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Rational Design of a Thermal Stable Variant of Human Carbonic Anhydrase II

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Title:
Rational Design of a Thermal Stable Variant of Human Carbonic Anhydrase II
Creator:
Boone, Christopher D
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (176 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Biochemistry and Molecular Biology (IDP)
Committee Chair:
MCKENNA,ROBERT
Committee Co-Chair:
BLOOM,LINDA B
Committee Members:
FLANEGAN,JAMES B
FROST,SUSAN COOKE
SILVERMAN,DAVID N
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Active sites ( jstor )
Biochemistry ( jstor )
Catalytic activity ( jstor )
Crystal structure ( jstor )
Disulfides ( jstor )
Enzymes ( jstor )
Molecules ( jstor )
Protons ( jstor )
Solar X rays ( jstor )
Thermal stability ( jstor )
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
crystallography -- dsc -- kinetics -- protein -- thermalstability
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
Human carbonic anhydrase II (HCA II) is a zinc-containing metalloenzyme that catalyzes the reversible hydration/dehydration of carbon dioxide into bicarbonate and a proton. Famous for its characteristically high catalytic turnover rate (106 s-1), HCA II has been of recent biomedical and industrial interest for implementation into carbon sequestration systems including artificial lungs and in bioremediation applications derived from the result of burning fossil fuels. However, the relative instability of HCA II in these environments (i.e., an acidic pH and temperatures in excess of 70 C) detrimentally affects the catalytic and overall cost efficiency of the system. These studies aim to rationally design a thermal stable variant of HCA II (without lowering the characteristic high catalytic efficiency of the enzyme) as to better withstand the aforementioned harsh industrial conditions. The proposed thermal stabilization mechanism involves site-directed mutagenesis of various sites in HCA II to include previously proposed stabilizing elements such as the reduction of surface hydrophobicity, engineering of disulfide bridges, incorporation of an aromatic cluster in the enzyme core, rigidification of surface loops via introduction of proline residues as well as deletion as surface loops. These variants of HCA II were measured for thermal stability in a variety of conditions utilizing differential scanning calorimetry and visualized via X-ray crystallography. Finally, the catalytic activities of the HCA II variants were measured using 18O mass spectrometry. The results showed that the most thermal stabilizing elements included surface reduction of hydrophobic residues and the inclusion of a conserved disulfide bridge. Combination of these two elements led to a dramatically thermal stabilized variant of HCA II (~20 C increase in melting temperature) with comparable catalytic activity to the wild-type enzyme. This variant is an excellent candidate for biomedical and industrial applications as it is not only very stable with good activity, but it also can be expressed in large quantities and is highly soluble in solution. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: MCKENNA,ROBERT.
Local:
Co-adviser: BLOOM,LINDA B.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31
Statement of Responsibility:
by Christopher D Boone.

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5/31/2015
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LD1780 2014 ( lcc )

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RATIONAL DESIGN OF A THERMAL STABLE VARIANT OF HUMAN CARBONIC ANHYDRASE II By CHRISTOPHER DANIEL BOONE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENT S FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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2014 Christopher Daniel Boone

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To whom it may concern

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4 ACKNOWLEDGMENTS I would like to thank Dr. Robert McKenna for his continual g uidance and encouragement during my doctoral candidacy. I would also like to thank the members of my committee: Dr. Linda Bloom, Dr. James Flanegan, Dr. Susan Frost and Dr. David Silverman for their advice and critiques towards my research. I thank Dr. Chi ngkuang Tu for teaching me how to perform and interpret the kinetic assays. I would also like to thank my outstanding undergraduate researchers Sonika Gill, Andrew Habibzadegan and Valerio Rasi for performing all of the experimental work. I thank Dr. Bala Venkatakrishnan for teaching me how to perform differential scanning calorimetry, Dr. Mayank Aggarwal and Dr. Art Robbins for teaching me how to grow, diffract and analyze X ray crystallographic data. I would also like to thank all of my co authors in whic h I have published: Dr. Mavis Agbandje McKenna, Dr. Shya Biswas, Dr. Zo Fisher, Barghav Kondeti, Melissa Pinard and Brittany Rife. I also thank the University of Florida, College of Medicine, and the Interdisciplinary Program, D epartment of Biochemistry & Molecular Biology for their acceptance into the program and for financial security. I also thank the entire McKenna lab family for critical critiques into my research and presentations. Finally, I would like to thank my mother, Vera Elizabeth Boone, and m y father, Billy Wray Boone, for their everlasting love and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ 4 LIST OF TABLES ................................ ................................ ................................ ........... 9 LIST OF FIGURES ................................ ................................ ................................ ...... 10 LIST OF ABBREVIATIONS ................................ ................................ .......................... 12 ABSTRACT ................................ ................................ ................................ .................. 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ... 16 1.1 Overview of the Carbonic Anhydrases ................................ ............................. 16 1.2 Structure of the Active Site ................................ ................................ .............. 16 1.3 Catalytic Mechanism ................................ ................................ ........................ 18 1.3.1 Overview ................................ ................................ ................................ 18 1.3.2 CO 2 Binding ................................ ................................ ........................... 19 1.3.3 Bicarbonate Coordination ................................ ................................ ....... 20 1.3.4 Proton Transfer ................................ ................................ ...................... 22 2 BIOENGINEERING OF CARBONIC ANHYDRASES ................................ ............ 26 2.1 Biomedical Applic ations ................................ ................................ ................... 26 2.1.1 Overview ................................ ................................ ................................ 26 2.1.2 Artificial Lungs ................................ ................................ ........................ 26 2.1.3 Blood Substit utes ................................ ................................ ................... 27 2.1.4 Antidote Delivery ................................ ................................ .................... 28 2.2 Industrial Usage ................................ ................................ ............................... 29 2.2.1 Overview ................................ ................................ ................................ 29 2.2.2 Atmospheric CO 2 Sequestration ................................ ............................. 29 2.2.3 Biofuel and Biomass Production ................................ ............................. 31 2.2.4 Integration of CO 2 Sequestration Systems ................................ ............. 32 3 RATIONAL STABILIZATION OF HUMAN CARBONIC ANHYDRASE II ................ 37 3.1 Overview ................................ ................................ ................................ ......... 37 3.2 Construction of a Thermophilic HCA II Variant ................................ ................. 38 3.2.1 Reduction of Hydrophobic Residues on the Protein Surface .................. 39 3.2.2 Engineering of Disulfide Bridges ................................ ............................. 40 3.2.3 Incorporation of Aromatic Ring Clusters ................................ ................. 40 3.2.4 Introduction of Proline Residues ................................ ............................. 41 3.2.5 Truncation of Flexible Surface Loops ................................ ..................... 41

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6 3.3 Combination of Stabilizing Elements ................................ ................................ 42 4 REDUCTION OF SURFACE HYDROPHOBICITY ................................ ................. 48 4.1 Overview ................................ ................................ ................................ ......... 48 4.2 Methodology ................................ ................................ ................................ .... 48 4.2.1 Protein Expression and Purification ................................ ........................ 48 4.2.2 X ray Crystallography ................................ ................................ ............. 49 4.2.3 Differential Scanning Calorimetry ................................ ........................... 50 4.2.4 Kinetic Studies ................................ ................................ ....................... 51 4.3 Results & Discussion ................................ ................................ ....................... 53 4.3.1 X ray Crystallography ................................ ................................ ............. 53 4.3.2 Stability ................................ ................................ ................................ .. 56 4.3.3 Catalytic Activity ................................ ................................ ..................... 57 4.4 Discussion ................................ ................................ ................................ ....... 58 4.6 Conclusions ................................ ................................ ................................ ..... 61 5 ENGINEERING OF DISULFIDE BRIDGES ................................ ........................... 68 5.1 Overview ................................ ................................ ................................ ......... 68 5.2 Methodology ................................ ................................ ................................ .... 69 5.2.1 Enzyme Expression and Purification ................................ ...................... 69 5.2.2 Crystallization and Diffraction Data Collection ................................ ........ 70 5.2.3 Differential Scanning Calorimetry ................................ ........................... 71 5.2.4 Catalytic Activity ................................ ................................ ..................... 71 5.3 Results ................................ ................................ ................................ ............ 72 5.3.1 X ray Crystallographic structure of DS1 ................................ .................. 72 5.3.2 Thermal Stability of Engineered Disufide Linkages ................................ 72 5.3.3 Catalytic Activity ................................ ................................ ..................... 74 5.4 Discussion ................................ ................................ ................................ ....... 75 5.5 Conclusions ................................ ................................ ................................ ..... 78 6 ADDITION OF AROMATIC CLUSTERS ................................ ................................ 84 6.1 Overview ................................ ................................ ................................ ......... 84 6.2 Methodology ................................ ................................ ................................ .... 84 6.2.1 Protein Expression and Purification ................................ ........................ 84 6.2.2 X ray C rystallography ................................ ................................ ............. 85 6.2.2 Differential Scanning Calorimetry ................................ ........................... 86 6.2.3 Kinetic Studies ................................ ................................ ....................... 86 6.3 Results ................................ ................................ ................................ ............ 86 6.3.1 X ray Crystallography ................................ ................................ ............. 86 6.3.2 Thermal Stability ................................ ................................ ..................... 87 6.3.3 Catalytic Activity ................................ ................................ ..................... 89 6.4 Discussion ................................ ................................ ................................ ....... 90 6.5 Conclusions ................................ ................................ ................................ ..... 91

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7 7 INTRODUCTION OF PROLINE RESIDUES ................................ .......................... 98 7.1 Overview ................................ ................................ ................................ ......... 98 7.2 Methodology ................................ ................................ ................................ .... 98 7.2.1 Protein Expression and Purification ................................ ........................ 98 7.2.2 X ray Crystallography ................................ ................................ ............. 99 7.2.3 Differential Scanning Calorimetry ................................ ......................... 100 7.2.4 Catalytic Activity ................................ ................................ ................... 100 7.3 Results ................................ ................................ ................................ .......... 100 7.3.1 X ray Crystallography ................................ ................................ ........... 100 7.3.2 Thermal Stability ................................ ................................ ................... 101 7.3.3 Kinetics ................................ ................................ ................................ 101 7.4 Discussion ................................ ................................ ................................ ..... 102 7.5 Conclusions ................................ ................................ ................................ ... 103 8 TRUNCATION OF SURFACE LOOPS ................................ ................................ 110 8.1 Overview ................................ ................................ ................................ ....... 110 8.2 Methodology ................................ ................................ ................................ .. 111 8.2.1 Protein Expression and Purification ................................ ...................... 111 8.2.2 X ray Crystallo graphy ................................ ................................ ........... 111 8.2.3 Differential Scanning Calorimetry ................................ ......................... 112 8.2.4 Catalytic Activity ................................ ................................ ................... 112 8.3 Results ................................ ................................ ................................ .......... 112 8.3.1 X ray Crystallography ................................ ................................ ........... 112 8.3.2 Thermal Stability ................................ ................................ ................... 114 8.3.3 Kinetics ................................ ................................ ................................ 114 8.4 Discussion ................................ ................................ ................................ ..... 115 8.5 Conclusions ................................ ................................ ................................ ... 116 9 COMBINATION OF STABILIZATION ELEMENTS ................................ .............. 124 9.1 Overview ................................ ................................ ................................ ....... 124 9.2 Methodology ................................ ................................ ................................ .. 124 9.2.1 Protein Expression and Purification ................................ ...................... 124 9.2.2 X ray Crystallography ................................ ................................ ........... 12 5 9.2.3 Differential Scanning Calorimetry ................................ ......................... 126 9.2.4 Catalytic Activity ................................ ................................ ................... 126 9.3 Results ................................ ................................ ................................ .......... 127 9.3.1 X ray Crystallography ................................ ................................ ........... 127 9.3.2 Thermal Stability ................................ ................................ ................... 128 9.3.3 Catalytic Activity ................................ ................................ ................... 129 9.4 Discussion ................................ ................................ ................................ ..... 130 9.5 Conclusions ................................ ................................ ................................ ... 133 10 CONCLUSIONS ................................ ................................ ................................ .. 140

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8 APPENDIX HORMONAL INHIBITION OF HCA II VIA THE BILE ACID CHOLATE 143 A.1 Overview ................................ ................................ ................................ ....... 143 A.2 Methodology ................................ ................................ ................................ .. 144 A.2.1 Enzyme Expression and Purification ................................ .................... 144 A.2.2 Crystallization and Diffraction Data Collection ................................ ...... 145 A.2.3 Inhibition Studies ................................ ................................ .................. 146 A.3 Results ................................ ................................ ................................ .......... 147 A.4 Discussion ................................ ................................ ................................ ..... 149 A.4 Conclusions ................................ ................................ ................................ .. 150 REFERENCES ................................ ................................ ................................ .......... 154 BIOGRAPHICAL SKETCH ................................ ................................ ......................... 176

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9 LIST OF TABLES Table page 4 1 X ray crystallographic data set and refinement statistics for the TS HCA II variants. ................................ ................................ ................................ ............ 62 4 2 Thermal stability and catalytic m easure ments of the TS HCA II variants ........... 63 5 1 X ray crystallographic data set and refin ement statistics for DS1 HCA II ........... 79 5 2 Thermal stability and catalytic m easurements for DS1 an d DS2 HCA II ............ 80 6 1 X ray crystallographic data set and refinement statistics for Phe226 HCA II Variants ................................ ................................ ................................ ............. 93 6 2 Thermal stability and catalytic m easurements of the Phe226 and Aros HCA II v ariants ................................ ................................ ................................ ............. 94 7 1 X ray crystallographic data set and refinement statistics for E234P HCA II ..... 105 7 2 Thermal stability and catalytic m easurements for E234P and K170P HCA II ... 106 8 1 X ray crystallographic data set and refinement statistics for 230 240 HCA II. 118 8 2 Thermal stability and catalytic m easurements for 230 240 HCA II. ................ 119 9 1 X ray crystallographic data set and refinement statistics for TS1/DS1 and TS2/DS1 HCA II ................................ ................................ .............................. 135 9 2 Thermal stability and catalytic m easurements of the TS1/DS1 and TS2/DS1 HCA II variants. ................................ ................................ ............................... 136 A 1 X ray cry stallographic data set and refinement statistics for HCA II/Cholate .... 151

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10 LIST OF FIGURES Figure page 1 1 Structural annotation of CAs ................................ ................................ .............. 24 1 2 Comparison of HCA II active sites with en hanced catalytic turnover rates ......... 25 2 1 Schemati c of the artificial lung system ................................ ............................... 34 2 2 Imm obilization of HCA II onto HFMs ................................ ................................ .. 35 2 3 Schematic of a HCA II carbon sequest ration system ................................ ......... 36 3 1 Reduction of s urface hydrophobicity on HCA II ................................ ................. 43 3 2 Proposed sites for the engineering of disulfide bridges in HCA II. ...................... 44 3 3 Proposed mutation sites for the creation of an aromatic cluster in HCA II. ......... 45 3 4 Proposed proline substitution sites on HCA II ................................ .................... 46 3 5 Proposed loop truncation site in HCA II ................................ ............................. 47 4 1 X ray crystallographic model of the TS HCA II variants ................................ ..... 64 4 2 Active site comparis o n of stabilized HCA II variants ................................ .......... 65 4 3 The rmograms for TS HCA II v ariants ................................ ................................ 66 4 4 Catalytic A ctivities of TS HCA II v ariants ................................ ........................... 67 5 1 X ray cryst allographic model of DS1 HCA II ................................ ...................... 81 5 2 Thermograms fo r DS1, DS2 and wild type HCA II ................................ ............. 82 5 3 Catalytic Activity of the DS1 and DS2 HCA II variants ................................ ....... 83 6 1 Overlay of the X ray crystallographic st ructures of the Phe226 variants ............ 95 6 2 Thermograms for the Phe226 and Aros HCA II variants ................................ .... 96 6 3 Catalytic activity of th e F226 and Aros HCA II variants ................................ ...... 97 7 1 Crystallogr aphic structure of E234P HCA II ................................ .................... 107 7 2 Thermograms of A) E234P and B) K170P HCA II ................................ ........... 108 7 3 Catalyti c activ ities of E234P and K170P HCA II ................................ .............. 109

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11 8 1 Crystallographic structure of 230 240 HCA II ................................ ................ 120 8 2 Thermogram for 230 240 HCA II ................................ ................................ ... 121 8 3 Catalytic activity of 230 240 HCA II ................................ ............................... 122 8 4 Schematic of the proton transfer water n etwork ................................ ............... 123 9 1 Global structure of the TS1/DS1 and TS2/DS1 HCA II variants ....................... 137 9 2 Thermograms of TS1/DS1 and T S2/DS1 HCA II at pH 7.8 and 5.6 ................. 138 9 3 Catalytic activity of the TS1/ DS1 and TS2/DS1 HCA II variants ...................... 139 A 1 X ray crystallographic structure of cholate binding to HCA II ........................... 152 A 2 Determination of I 50 from catalytic activity ................................ ........................ 153

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12 LIST OF ABBREVIATIONS Aros HCA II L47F/V49F/I146F/L212F HCA II BA Bile Acid CA Carbonic Anhydrase CAI Carbonic Anhydrase Inhibi tor CaCO 3 Calcite CAT Catalase CO 2 Carbon Dioxide DSC Differential Scanning Calorimetry D w Deep Water DS1 HCA II A23C/L203C/C206S HCA II DS2 HCA II G6C/N11C/A23C/L203C/C206S HCA II HCA Human Carbonic Anhydrase HCA II Human Carbonic Anhydrase isofo rm II HCA IV Human Carbonic Anhydrase isoform IV HCA IX Human Carbonic Anhydrase isoform IX HCO 3 Bicarbonate HFM Hollow Fiber Membrane k B Rate of proton transfer k cat /K M Catalytic efficiency MgCO 3 Magnesite ppm Parts Per Million polySFHb 3 5 stro ma free hemoglobin molecules SOD Superoxide Dismutase TS1 HCA II L100H/L224S/L240P HCA II

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13 TLM Thin layer membranes TS2 HCA II Y7F/L100H/L224S/L240P HCA II TS3 HCA II Y7F/N62L/L100H/L224S/L240P HCA II TS4 HCA II Y7F/N67Q/L100H/L224S/L240P HCA II TS5 HCA II Y7F/N62L/N67L/L100H/L224S/L240P HCA II W# Water molecule # ZnH 2 O Zinc bound water ZnOH Zinc bound hydroxide SspCA Sulfurihydrogenibium yellowstonense YO3AOP1 CA 240 HCA II HCA II with residues 230 240 deleted

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14 Abstract of Dissertati on Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RATIONAL DESIGN OF A THERMAL STABLE VARIANT OF HUMAN CARBONIC ANHYDRASE II By Christopher Da niel Boone May 2014 Chair: Robert McKenna Major: Medical Sciences Human carbonic anhydrase II (HCA II) is a zinc containing metalloenzyme that catalyzes the reversible hydration/dehydration of carbon dioxide into bicarbonate and a proton. Famous for its characteristically high catalytic turnover rate (10 6 s 1 ), HCA II has been of recent biomedical and industrial interest for implementation into carbon sequestration systems including artificial lungs and in bioremediation applications derived from the res ult of burning fossil fuels. However, the relative instability of HCA II in these environments (i.e., an acidic pH and temperatures in excess of 70 C) detrimentally affects the catalytic and overall cost efficiency of the system. These studies aim to rati onally design a thermal stable variant of HCA II (without lowering the characteristic high catalytic efficiency of the enzyme) as to better withstand the aforementioned harsh industrial conditions. The proposed thermal stabilization mechanism involves site directed mutagenesis of various sites in HCA II to include previously proposed stabilizing elements such as the reduction of surface hydrophobicity, engineering of disulfide bridges, incorporation of an arom atic cluster in the enzyme core and rigidificati on of the enzyme surface loops via introduction of proline residues in key location or truncation of the loops These variants of HCA II were

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15 measured for thermal stability in a variety of conditions utilizing differential scanning calorimetry and visualiz ed via X ray crystallography. Finally, the catalytic activities of the HCA II variants were measured using 18 O mass spectrometry. The results showed that the most thermal stabilizing elements included surface reduction of hydrophobic residues and the inclu sion of a conserved disulfide bridge. Combination of these two elements led to a dramatically thermal stabilized variant of HCA II (~20 C increase in melting temperature) with comparable catalytic activity to the wild type enzyme. This variant is an excel lent candidate for biomedical and industrial applications as it is not only very stable with good activity, but it also can be expressed in large quantities and is highly soluble in solution.

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16 CHAPTER 1 INTRODUCTION 1.1 Overview of the Carbonic Anhydrases The carbonic anhydrases (CAs) are a family of mostly zinc containing metalloenzymes that catalyze the reversible hydration of CO 2 to bicarbonate and a proton ( 1 3 ) There are five evolutionarily distinct classes of C A class expressed in mammals ( 4 ) ( 5 ) ; ( 6 7 ) ; which were first discovered in marine diatoms ( 8 9 ) Humans express 15 isoforms of C A s (three of which are acatalytic) which are ubiquitously found throughout the body ( 10 11 ) with human CA isoform II (HCA II) being the best studied. HCA II is a 29 kDa monomeric cytosolic enyzme ( Figure 1 1A ) that is expressed in a variety of systems including inside of erythrocytes, along the gastrointestinal tract an d in the kidneys ( 11 ) 1.2 Structure of the Active Site CAs, including most HCA isoforms, reveal that the catalytic site is located deep within the protein structure and accessible to s olvent by a large open conical cleft that is approximately 15 in depth and width (volume of ~900 3 ) ( 1 4 12 13 ) The Zn 2+ metal at the base of the active site is in a distorted tetrahedral coordination with three His residues ( numbered 92, 94 and 119) and a wat er molecule ( Figure 1 1B ). Proper orientation of the imidazole rings to allow for correct coordination of the metal ion occurs via several hydrogen bond interactions including : His119 His107 ( 14 )

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17 The catalytic site is separated into hydrophobic and hydrophilic faces with the hydrophobic portion being further categorized into two topological locations : the residues which comprise the CO 2 binding site and those resid ues which line the entrance of the active site ( Figure 1 1B ). These proximal (residues 121, 143, 198, 207 and 209) and distal (residues 131, 135, 201, 202 and 204) hydrophobic residues relative to the Zn 2+ metal are often referred to as the primary and sec ondary hydrophobic binding sites, respectively ( 3 4 1 5 ) The secondary hydrophobic binding site is important in facilitation of CO 2 into the active site and defining the binding affinities of several CA inhibitors (CAIs) including the anti glaucama therapeutic agents aceta zolamide and methazolamide ( 16 19 ) The hydrophilic residues of the binding cavity (residues 62, 64, 67, 92, 106, 199 C A s and can be subdivided according to their role in catalysis ( 20 ) Glu106 which properly aligns Thr199 to acc ept a hydrogen bond from the Zn OH and orients t he lone electron pair of the Zn OH for nucleophilic attack on CO 2 (Eq. 1 1) ( 4 12 14 21 22 ) se veral CAIs that displace the Zn OH ( 17 ) Tyr7, Asn62, Asn67 and Thr200 stabilize the ordered water network that transfers the proton produced during catalysis (Eq. 1 2 ) out of the active site via the proto n shuttle residue His64 ( Figure 1 1B ) ( 20 23 24 ) X ray and neutron crystallographic models of C A s over t he past 20 years in various pH and buffer conditions reveal that two rotamers of His64 exist, commonly 2+ metal ( 4 20 25 26 ) There are theories that proton transfer from His64 into the bulk solve nt arise from either

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18 a flipping mechanism ( 20 ) or tautomerization of the histidine ring ( 27 ) His64 has been observed in two stat es, an outward configuration at pH 5.7, presumably in a protonated state, whereas in basic conditions it is seen in the inward conformation and deprotonated ( 12 14 26 28 31 ) The pK a of His64 has been shown to be sensitive to the surrounding residues and can have an e ffect on catalytic rates ( 32 37 ) 1.3 Catalytic Mechanism 1.3.1 Overview The mechanism of catalytic conversion of carbon dioxide into bicarbonate and a proton by HCA II is believed to be shared by all CAs ( 24 38 ) HCA II is one of the fastest enzymes known, with a catalytic turnover (k cat ) of ~1 s 1 and maximum catalytic efficiency (k cat /K M ) approaching the diffusion limit at 100 M 1 s 1 ( 1 3 ) Catalysis occurs via a two step ping pong mechanism: H 2 O CO 2 + EZnOH EZnHCO 3 EZnH 2 O + HCO 3 (1 1) EZnH 2 O + B EZnOH + BH + (1 2) The binding of CO 2 (E q. 1 1) in the primary hydrophobic binding site of the HCA II active site promotes the nucleophilic attack by the zinc bound hydroxide (ZnOH ) leading to the formation of bicarbonate (HCO 3 ), which is later evacuated from the active site via the random diffusion of water into the active sit e. The transfer of a proton in E q. 1 2 f rom the bound water molecule at the zinc (ZnH 2 O) to an acceptor in the bulk solvent (B) is needed to regenerate the h ydroxide for a subsequent round of catalysis via the proton shuttle residue His64 ( 23 24 38 39 ) This transfer occurs on the order of 10 6 s 1 for HCA II and is the rate limiting step of the overall maximum velocity of catalysis ( 24 40 ) The intramolecular proton transport between the ZnH 2 O and His64

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19 occurs via intervening water molecules in the active site. From His64, an in termolecular transfer event delivers the proton to the bulk solvent. 1.3.2 CO 2 Binding Early studies on the binding of CO 2 relied on the use of substrate analogs and inhibitors due to high catalytic activity of HCA II ( 41 ) Crystallographic studies of HCA II in the presence of these same substrate analogs and inhibitors reveal that sulfonamide inhibitors such as acetazolamide displace the ZnOH keeping the same tetrahedral coordination with the Zn 2+ metal. Structures of HCA II with anionic inhibitors, such as cyanate, revealed the displacement of the deep water (D W ) molecule, which is coordinated by the amide nitrogen of Thr199, to create a new trigonal bipyramidal coordination sphere of the Zn 2+ metal ( 42 ) Furthermore, spectroscopic studies indicated that the binding of CO 2 does not require any inner sphere coordination by the Zn 2+ metal ( 43 45 ) These results ultimately led to the conclusion that CO 2 must interact with residues in the prim ary hydrophobic binding site and that Thr199 plays a specific role in CO 2 orientation ( 46 52 ) Molecular dynamic simulations revealed that the primary binding site was not the only binding site of CO 2 but that there are secondary and tertiary sites in the active site that are suggested to play in a role in rapid diffusion of CO 2 into the catalytic site ( 22 53 ) The binding of CO 2 in the active site of CA is weak (K d 100 mM) and even lower for bicarbonate ( 3 45 ) The X ray crystallographic structure of HCA II in complex with CO 2 ( Figure 1 1C ) was obtained via high pressure pumping of room temperature CO 2 inside of a cryo cooling apparatus above a liquid nitrogen bath ( 54 56 ) The crystals w ere dipped slowly into the liquid nitrogen bath over a two minute span to trap CO 2 in the active site ( 52 ) The high resolution (1.1 ) CO 2 bound HCA II structure revealed that the backbone

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20 amide of Thr199 could make a potential hydrogen bond with CO 2 at a distance of 3.5 . The orientation of CO 2 via Thr199 is such that the oxygens of the subst rate are equidistant from the zinc bound solvent molecule (3.0 and 3.1 ) while the carbon atom is 2.8 away. However, the Zn 2+ metal only seems to play a minor role in interacting with and orientating the CO 2 T he structure of the substrate bound, apo HCA II revealed that CO 2 undergoes a minor rotation about its long axis towards the uncoordinated histidine residues that bind metal in the holo enzyme. A s a result the oxygen atom in CO 2 moves closer towards the amide nitrogen of Thr199 (3.1 versus 3.5 for apo and holo HCA II, respectively). 1.3.3 Bicarbonate Coordination The first structure of bicarbonate bound in th e active site of HCA II ( Figure 1 1C ) was achieved with the variant T200H which has a higher binding affinity for bicarbonate than the native enzyme ( 57 ) The structure revealed that one of the oxygens of the bicarbonate has the same spatial positioning as the zinc bound solvent molecule seen in other structures and the other two oxygens of the bicarbonate are shifted about 1.2 away from the coordinates seen of the CO 2 bound structure. Later studies with the mutant T199P/C206S of HCA II revealed that the T200H mutation had minimal effect on the positioning of the bicarbonate ( 58 ) Bi carbonate was first seen in crystallographic models of native HCA II as the result of enzyme activation via X ray damage from either water photoradiolysis and/or electron radiolysis ( 55 ) The model showed that O3 of bicarbonate is bound 2.0 away from the Zn 2+ metal and is within hydrogen bond distance of the hydroxyl group of Thr199 (2.6 ) whereas the O2 atom of bicarbonate makes a hydrogen bond with the backbone amide of Thr199 (2.9 ). The O1 atom of bicarbonate is 2.9 away from the

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21 Zn 2+ ion. Like CO 2 bicarbonate was observed to make van der Waals contacts with the residues lining the hydrophobic pocket (Val121, Val143, Leu198 and Trp209). A crystallographic structure of both CO 2 and bicarbonate bound in the active site of HCA II with the mutation V143I confirmed that the oxygens of the bicarbonate mimic the coordinates and geometry seen with the oxygens of the CO 2 the zinc bound solvent molecule and prev ious bicarbonate bound structures ( Figure 1 1C ) ( 59 ) The authors note that the V143I mutation has moved the CO 2 closer to the zinc metal by 0.3 and lowered the k cat /K M by ~15 fold as compared to wild type HCA II. The differences seen in the position of the bicarbonate molecule in the V143I variant to that of other bicarbonate bound HCA II structures could be due to the mutation itself but also could be an artifact of diffusing bicarbonate into crystal s instead of capturing bicarbonate formation immediately after catalysis. The structures of all substrate and product bound CAs correlate well with the proposed catalytic mechanism of CAs which is that of direct nucleophilic attack of the ZnOH on CO 2 Formation of bicarbonate promotes a bidentate interaction with the zinc metal, resulting in a penta coordinated metal ion with trigonal bipyramidal geometry. A water molecule displaces the zinc bound bicarbonate which is then deprotonated via the proton tr ansfer water network for the subsequent round of catalysis. A previously unidentified water molecule was observed in both the apo and holo CO 2 bound HCA II structures which may play the role of displacing bicarbonate ( 52 54 ) This water molecule is located between the side chain of Thr200 and the CO 2 molecule and could allow for easier access to the bicarbonate molec ule and increased catalytic activity (as compared to random diffusion of water from the bulk solvent into the active site).

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22 1.3.4 Proton Transfer The neutron crystallographic structures of HCA II at pH 7.8 ( 60 ) pH 10.0 ( 29 ) and in complex with the clinically used inhibitor acetazolamide ( 61 ) has allowed for t he observance of the ZnOH with explicit information on hydrogen atom positions and their dependence on pH ( 25 ) Moreover, observation of the hydrogen positions showed the ionization state s of the imidazole ring of the proton shut tle His64 and other residues near the active site, such as Tyr7 were unionized at pH 7.8 ( Figure 1 1B ). T he orientation of these hydrogen bonds was not appropriate for proton transfer at high pH ; that is, breaking of hydrogen bonds and reorientation of wa ter mol ecules would be necessary for proton transfer Neutron crystal structures subsequently determined at ambient pH showed a reorganization of the hydrogen bonded water network more consistent with proton transfer Such reorganization steps probably con tribute significantly to the observed free energy barrier to proton transfer Computational studies have elucidated a number of possible proton pathways in HCA II between the ZnOH His64 and external solution ( 62 ) Molecular simulation studies were consistent with a model for proton motion from ZnOH to an Eigen cation (H 9 O 4 + ) on W1, which is then transferred onto a Zundel cation (H 5 O 2 + ) containing W2, and then onto another Eigen cation on W3A and finally to His64 ( 63 64 ) (See Figure 1B for labeling of water molecules.) The barrier making the largest contribution to the rate is a high free energy Eigen cation formed by the excess proton residing on W3A with a sma ll amplitude contributio n from W 2 and His64. This establishes a barrier with a free energy of 10.0 kcal mol 1 and a rate constant for proton transfer near 1.0 s 1 Water structures with more than three water molecules represent clusters that may be branched, and therefore are more likely to form an Eigen cation which has been

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23 shown to elevate the proton transfer barrier when compared to smaller unbranched wate r wires ( 65 ) The se results indicate that the Y7F variant of HCA II possesses a significantly elevated probability of forming smaller water clusters of size 3 ( Figure 1 2A) ; this favors an unbranched water structure and a higher probability of proton transfer that is likely to account for the elevated turnover number of 4 to 7 s 1 reported by kinetic studies ( 66 ) Wild type HCA II w ith a turnover number near 1 s 1 favors water clusters of size 4 which is more likely to involve an Eigen cation and hence a higher overall barrier to proton transfer. Additionally, Y7F exhibits lifetimes (5.0 ps) for water structure g reater than twice t hat of the wild type water wires. Part of the explanation for these longer lifetimes of specific water structures may be that the Y7F variant can expand and contract without placing strain on the water wire responsible for proton transport ( 66 67 ) These computational studies have offered a strong complementary approach to models based on observations in solution and in crystal structures. In addition to the Y7F mutation, an HCA II variant that also contains the mutation N67Q has been shown to possess a turnover number 1 compared with a value 1 for wild type HCA II ( 34 ) This higher rate of proton transfer observed for Y7F N67Q HCA II could not be explained by differences in the values of the pK a of the proto n donor (His64) and acceptor ( Zn OH ) or by orientation of the side chain of the proton shuttle residue His64 but appears to be associated with reduced branching in the active site water networks as observed in crystal structures. Moreover, Y7F N67Q HCA II is unique among the variants studied in having a direct, hydrogen bonded chain of water molecules between the zinc ( Figure 1 2B) ( 34 )

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24 Figure 1 1. Structural annotation of CAs. A) The structure of HCA II (PDB: 3KS3; ( 68 ) ) shown in cartoon rep resentation with the C backbone colored in light blue the Zn 2+ metal ion as a pink sphe re and the coordinating histidine residues in sticks B) Structure of the active site (PDB: 3TMJ ; ( 29 ) ) showing the or dered water network (stick view ) hydrogen bond (dashed line) to their respective hydrophilic contacts (blue sticks). The hydrophobic residues involved in coordinating CO 2 in the active site are shown as orange sticks. The C backbone h as been omitted for clarity. (C) Stick models of the bicarbonate and CO 2 binding sites. Active site residues are colored and labeled as in (B).

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25 Figure 1 2. Comparison of HCA II active sites with enhanced catalytic turnover rates. A) Schematic of the wate r network (red spheres) in the Y7F HCA II variant (PDB: 2NXR; ( 32 ) ) shown as light orange sticks and the Zn 2+ metal as a magenta sphere. The zinc bound sulfate (SO 4 ) is shown in stick model. Hydrogen bond interactions are shown as black dashed lines. B) Schematic of the water network in the Y7F/N67Q HCA II variant (PDB: 4IDR; ( 34 ) ) shown in blue stick model.

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26 CHAPTER 2 BIOENGINEERING OF CARBONIC ANHYDRASES 2.1 Biomedical Applications 2.1 .1 Overview The successful immobilization of CA onto thin liquid membranes (TLMs) ( 69 ) has accelerated research into CO 2 capture for biomedical applications. The TLMs are typically comprised of polypropylene derivatives that act as semi permeable barrier, allowing for diffusion of CO 2 from a flowing gas outside the membrane into the aqueous layer located underneath. This aqueous layer contains CA which will catalyze the hydration of CO 2 into a highly more soluble form, HCO 3 which can then be subsequently desorbed by lowered CO 2 partial pre ssures further downstream ( 69 70 ) A major benefit of TLM based system is that they operate very efficiently at ambient pressures and temperatures, but they do also have some longevity concerns that will need to be addressed, such as keeping the membranes from drying and breaking, before their practical use is feasible ( 71 ) The need for efficient removal or detection of CO 2 in the biomedical field has grown recently in applications such as artificial lung systems or in antidote delivery has lead to the bioengineering of CAs for such purposes This chapter describes the use and production of kinetically enhanced and/or stabilized CA variants to aid in the development of cost efficient and productive CO 2 capture systems. 2.1 .2 Artificial Lungs The same principles used in industrial TLM CO 2 sequestration hav e been extended for implementation into extracorporeal artificial lungs ( Figure 2 1) to facilitate blood gas exchange in re spiratory failure patients. However, further improvements in

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27 efficacy in these devices are needed before becoming an effective altern ative treatment to mechanical ventilators ( 72 75 ) Current limitations of artificial lung systems include the inefficient transfer of CO 2 (from a blood inlet) acro ss the polymetric hollow fiber membrane (HFM) where it can then be flushed out of the system by a stream of oxygen ( 76 77 ) A large surface interface (1 2 m 2 ) composed primarily of various cross linked saccharides, is required for sufficient gas exchange, which leads to issues with hemocompatibility and biocompatibility ( 78 83 ) The transfer of CO 2 across the membrane can be accelerated via immobilization of CA onto the surface of the HFM ( Figure 2 2) thereby reducing the required surface area for an effective gas exchange rate ( 76 77 84 ) monstrated a 75% increase in the rate of CO 2 removal compared to an untreated HFM. Immobilization occurs via either isouruea or N substituted imidocarbonate covalent linkages between surface amine groups of CA and cyanate esters or cyclic imidocarbonates o n the surface of the HFM. This technology has been coupled with impeller devices to increase the rate of blood mixing and CO 2 transfer across the HFM, but the increased shear forces denatured the immobilized CA, leading to a loss of enzyme function ( 76 ) 2.1.3 Blood Substitutes In contrast to artificial lung systems where CAs are used to capture CO 2 for extraction from the blood, there have also been studies done that utilized the same for carbon capture in blood substitutes ( 85 ) These alternative blood supplies are primarily composed of four to five cross linked stroma free hemoglobin molecules (termed polySFHb) that have been shown to be advantageous over transfused whole blood because they can be autoclaved, have long storage capabilities and contain no blood antigens ( 86 ) These polySFHb substitutes, however, dis played inadequate CO 2

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28 removal rates. As a continual source of blood is needed for surgical use and natural blood is often limited in supply, research has been devoted to improvement in these blood substitutes. The engineering of superoxide dismutase (SOD), catalase (CAT) and CA enyzmes into the blood substitutes (PolySFHb SOD CAT CA) displayed encouraging carbon capturing capabilites and antioxidant properties ( 85 ) 2.1.4 Antidote Delivery CAs have also been employed in the pharmacology field as CO 2 sensors involved in ant idote delivery systems used in the treatment of analgesic overdose ( 87 ) Other medicines that have very potent analgesic effects include the opioids but overdoses can cause respiratory hypoventilation which lead to elevated somatic CO 2 levels and to an acidosis induced death. Monitoring of blood opioid levels with morphine activated enzymes that release the antidotes naltrexone and naloxone, which ha ve been pre packaged into polymer clathrates, have shown to be a successful responsive system ( 88 ) An alternative treatment for analgesic overdose which does not depend on monitoring of the blood opioid levels is the CA system which shares similar properties to that seen with other cation ic hydrogels such as c hitosan and alginate ( 89 90 ) It is unique, however, in that it reacts to a toxicity biomarker, such as high CO 2 levels or acidic pH, in an antidote feedback regulated manner. The hydrogel is composed of N,N dimethyaminoethyl methacrylate (DMAEMA) polymers that have been modified to have a pK a of ~7.5, making it an adequate blood pH monitor and has also been incorporated into a glucose sensitive insulin releasing system that included CA as a CO 2 sensor ( 91 ) Further research into the hydrogel design has lead to a switchable co block polymer that undergoes a reproducible transition from gel to sol u pon CO 2 exposure ( 92 ) This

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29 transition was utilized to trigger the CO 2 induced release of an encapsulated protein, extenuating future potenti al biomedical applications of a CA based drug delivery system that is sensitive to changes in CO 2 bicarbonate or pH. 2.2 Industrial Usage 2.2 .1 Overview The favorable properties of HCA II (high kinetic parameters, easy expression, high solubility, interme diate heat resistance) have made it an attractive candidate for numerous industrial applications including as a bio catalyst for carbon sequestration of flue gas from coal fired power plants ( 93 ) Also, there are established protocols utilizing CA found in algae to capture CO 2 and convert it into biofuels and other valuable products ( 94 95 ) There is also interest in using apo CAs as a bio sensor for zinc and other transition metals in sea water or human serum ( 96 ) For industrial applications, sm all improvements in stability without detriment to yield, activity or solubility, can accelerate the development of H CA II as a better bio catalyst. Use of the free enzyme in solution can also have disadvantages, as the low stability can limit recycling an d cost efficiency in an industrial setting ( 97 ) 2.2 .2 Atmospheric CO 2 Sequestration The increase in atmospheric concentrations of the greenhouse gases, including CO 2 methane, chlor oflurocarbon and nitrous oxide, has been associated with human induced activities ( 98 ) Of particular concern is the rise in the highest absorbing greenhouse gas, CO 2 since the pre industrial era (~1850), rising from ~280 ppm ( 99 ) to over 400 ppm in 2013 ( 100 ) Measurement of the CO 2 content from core extracts of Antarctic ice indicate that atmospheric concentrations are higher today than in the past 800,000 years ( 101 104 ) Other geological evidence, based on a boron isotope ratio in

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30 ancient plan ktonic foraminifer shells, suggests that comparable CO 2 atmospheric concentrations were last seen about 20 million years ago, during the first and longest warming period of the Miocene series ( 105 ) The burning o f fossil fuels has been associated with ~75% of the increase in atmospheric levels of CO 2 over the past 20 years, with the remainder primarily due to deforestation ( 106 ) S ince the post industrial era (1896) the continuing rise in atmospheric CO 2 concentrations has been correlated with an increase in global surface temperatures ( 107 ) Measurement of the average global temperature over a 100 year span (1906 2005) revealed an average inc rease by 0.7 0.2 C over that period, compared to the relatively stable temperature for the prior 2000 years ( 108 ) Associatively, elevated surface temperatures accelerates the melting of glacier and polar ice caps, leading to a rise in sea levels, ocean acidification and desalination; raising concerns over preservation of n umerous animal and plant species, and ecological systems ( 109 111 ) Selectively capturing CO 2 out of a mixture of waste flue gas (typically 10 20% CO 2 content) that also includes nitrogen, sulfurs and other org anic compounds can be expensive and technically challenging ( 112 113 ) Current industrial protocol employs indirect methods of CO 2 capture which begins with dissolution of the relatively insoluble CO 2 into an aqueous phase via amine scrubbing or mineral carbona tion. An attractive alternative includes the design and incorporation of an environmentally benign, renewable, selective and inexpensive biomietic CO 2 sequestrating agent ( Figure 2 3) The hydrated CO 2 either expressed as carbonic acid (H 2 CO 3 ) or as the c onjugate base, bicarbonate (HCO 3 ) (depending on pH), can then be chemically converted into the

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31 carbonates calcite (CaCO 3 ) or magnesite (MgCO 3 ), or other mineral derivatives (aragonite, vaterite) for industrial and agricultural purposes ( 114 ) The successful sequestration of CO 2 from industrial flue gas raises concerns for its long term storage. The seque stered gas can either be pressurized to a liquid or chemically converted to a stable compound, which can then be stored underground or in the ocean ( 112 115 ) Additionally, the production of calcite or magnesite with subsequent burial of the solid carbonates is actively being studied as a possible solution, but concerns over the effects of acid rain on these deposits have arisen d ue to the possible sudden release of CO 2 ( 116 ) The carbonates have low solubility in water and are extre mely durable, as evidenced by being a main constituent of shells in marine organisms ( 117 118 ) Calcite is al so a common component for various pigments, acid neutralizers and construction materials. Alternately, the captured CO 2 can be converted into various beneficial byproducts including stable storage polycarbonates, acrylates and methane ( 119 121 ) 2.2 .3 Biofuel and Biomass Production The limited availability of fossil fuel deposits and growing concerns for the long term global environmental effects over the burning o f these products has prompted many countries, including the U.S., to search for alternative fuel sources ( 122 ) In the U.S. alone, there are an estimated 60 billion gallons of diesel and 120 billion gallons of gasoline used for transportation every year ( 123 ) Accounting for gasoline being only ~65% as efficient as diesel, this equates to a total of ~140 billion gallons of fuel needed every year to satisfy consumer demand. Biodiesel is pre ferential over conventional diesel in that it emits less gaseous pollutants, including zero CO 2 and sulfate emission, into the atmosphere and is non toxic ( 94 ) However, only 15% of the U.S. biodiesel

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32 demand could be satisfied if all of the arable land in the U.S. were used to grow soybean for oil production (which accounts for over half of the U.S. source for biodiesel) ( 122 124 ) The current production of biofuels also displaces croplands and has been associated with increased consumer prices ( 125 126 ) An attractive alternative to the soybean derived production of biofuels are algae based systems. Compared to terrestrial plants, algae (cyanobacteria) have higher oil production and carbon fixation rates ( 127 128 ) Algae are an environmental friendly alternative as they would naturally sequester atmospheric CO 2 require only sunlight and minimum micronutrients for growth and they do not compe te with agricultural lands as they can be cultivated in ponds or enclosed photobioreactors located on non arable land ( 122 ) Additional medicinal agents and byproducts in which can be harvested from algal cultures include proteins, fatty acids, vitamins, mie nerals, pigments, dietary supplements and agents used in food production, fertilizers and other commodity products ( 129 131 ) The effects of CA on carbon flux and f ixation rates in algae have shown enhanced biomass production via addition of lysed endogenous cytoplamsic Dunaliela sp. CA to algal cultures ( 132 133 ) Ongoing research investigating the effect of adding engineered extracellular CAs to algal cultures should provide further advancement in biomass and biodiesel production ( 134 ) 2.2.4 Integration of CO 2 Sequestration Systems Similar to the atmospheric CO 2 seques tration tec hniques described in Section 2.2.2 algal and cyanobacteria cultures can be used to produce calcite indirectly, as evidenced in Ch lorella and Spirulina sp. and is an ongoing area of research ( 95 130 131 135 ) The natural precipitation of calcite in microalgae serves many proposed roles including buffering purposes and as a safegaurd against active transport of bicarbonate

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33 ions ( 135 ) Studies have been performed aime d to simultaneously enhance lipid production and CO 2 capture in algal cultures, with Chlorella sp. showing promising results ( 94 95 ) Along with the formation of calcite in these systems, additional evidence ( 95 ) for utilization of CA in bicarb onate production during these processes came with observed decreased CO 2 capture upon addition of acetazolamide, a tight binding inhibitor of the CAs ( 47 61 136 137 ) The catalytic activities of extra and intra cellular CA in red tide dinoflagellates have shown to be pH dependent, with one species displaying increased bicarbonate uptake at or above pH 9 ( 138 ) These environmentally dependent catalytic rates can be utilized in the design of an optimized system (for pH, nutrient availability, aeration, etc.) for the simultaneous production of biofuels and calcite in a cost efficient manner.

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34 Figure 2 1. Schematic of the artificial lung system Blood from the patient is flowed over the HFMs where the captured HCO 3 (blue diamonds) is catalyzed by HCA II to CO 2 (white rods). The gas then diffuses across the HFM where it is evacuated from the system in an oxygen stream.

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35 Figure 2 2. Immobilization of HCA II onto HFMs. Artist reconstruction of a covalent linkage between the C terminal lysine on the enzy me (shown in cartoon view) and the polymeric membrane. The Zn 2+ metal is shown as a magenta sphere. Coordinating residues and substrate molecules are shown in stick view.

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36 Figure 2 3. Schematic of a HCA II carbon sequestration system. Flue gas containi ng CO 2 produced from the burning of fossil fuels is dissolved in an aqueous solution and passed into a reactor containing the enzyme immobilized onto a polymeric bead. The dissolved CO 2 is then catalyzed into HCO 3 where is can undergo geosequestration or be converted into environmental friendly byproducts such as calcite.

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37 CHAPTER 3 RATIONAL STABILIZATION OF HUMAN CARBONIC ANHYDRASE II 3.1 Overview The rate limiting step in current biomedical and industrial carbon capture methods is the selective hydration of CO 2 warranting research into using CA s as a carbon sequestration catalyst ( 139 ) Human (and other mammalian) CAs offer several advantages as they are an extremely efficient and specific means for CO 2 capture, ar e easily overexpressed in bacteria or commercially available, are re usable, and operate at ambient temperatures and under mild conditions ( 140 141 ) However, curre nt utilization of HCA II in these applications is limited by the relative instability of the enzyme i n the harsh environment (e.g., organic solvents low pH and high temperature), resulting in an overall reduction in cost efficiency and productivity ( 93 97 142 ) Additionally, HCA II is irreversibly denatured at ~58 C ( 93 143 ) and is susceptible to inhibition by small anio ns including sulfate, cyanate, thiocyanate and azide ( 4 47 ) The production of therm al stabilized enzymes i s still a significant challenge and there are many approaches to this, each with varying success. One popular strategy is to create large libraries of mutants created through random mutagenesis and directed evolution while selecting for a specific criteria ( 144 ) Other studies have focused on a more rational approach with a small set of targeted changes, like introducing Arg residues as stabilizing elements ( 145 ) Many studies have shown protein stability to be a function of many aspects including protein folding, co re packing, surface electrostatics, to overall rigidity and it appears that these determinants have varying importance in different proteins ( 146 148 )

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38 Computa tional tools have been developed to deal with these uncertainities that can assist with rational thermostability design, but they do not, however, suggest a generalizable strategy that will work for all proteins ( 147 149 ) Another successful approach is known as the B FIT method where areas in a protein with high thermal fluctuations are identified from the X ray crystal structures and then are subjected to random mutagenesis to meet an energy minimum ( 150 ) The development of rational design approaches based on specific crystallographic data that inform on surface electrostatics, hydrophobic interactio ns, as well as hydration and hydrogen bonding are appealing because this can lead to the development of guidelines for thermostabilization of all proteins. However, due to the complex nature of protein folding, kinetics, and stability, the most effective s trategy will likely be a combination of many techniques, both computational and experimental. 3.2 Constructi on of a Thermophilic HCA II Variant The free energy of stabilization ( G) of a protein is the difference between the free energies of the folded a nd unfolded states and directly measures the thermodynamic stability of the protein. The stabilization enthalpy ( H) and entropy ( S) are large values that vary almost linearly with temperature within the activity range of the enzyme whereas G is usually small (5 15 kcal mol 1 ) ( 151 152 ) Surprisingly, there is not one single distinguishable trait that predetermi nes if a particular enzyme will be more thermal stable than an other. In fact, most thermal stable variants are very similar to their mesostable counterparts with 40 to 85% sequence identities ( 153 154 ) super imposable three dimensional structures ( 155 161 ) and displaying the same catalytic mechanisms ( 154 162 163 ) As a result, the G values of hyperthermophilic

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39 and mesophilic enzymes is small, usually in the range of 5 to 20 kcal mol 1 ( 164 ) Additionally, stability studies of enzyme mutants revealed that differences in G as small as 3 to 6.5 kcal mol 1 can account for thermostability increases up to 12 C ( 165 166 ) The studies outlined below will aim to create a variant of HCA II to maximize the G of unfolding without d etrimentally affecting its catalytic efficiency These proposed variants will be studied via differential scanning calorimetry for their stability 18 O exchange mass spectrometry for their catalytic properties and X ray crystallography for the structural c onsequences of these introduced mutations (see Section 4.2 for experimental details). 3.2.1 Reduction of Hydrophobic Residues on the Protein Surface The hydrophobic effect is considered to be the major driving force of protein folding and, hence, is the m ajor force responsible for protein stability ( 151 167 ) Hydrophobic residues found on the surface of proteins ar e detrimental to protein stability and solubility as they cannot participate in stabilizing interactions with the solvent (no enthalpic contributions) and the ordering of solvent molecules around these residues into clathrates decrease the overall entropy of the system. A number of hyperthermophilic proteins show significantly reduced hydrophobic accessible surface areas (ASA) with an average of ~10% less hydrophobic ASA compared to mesophilic homologs ( 167 ) In HCA II, there is a cluster of hydrophobic ASA posterior to the active site opening that is comprised of three leucine resid ues (Leu100, Leu224 and Leu240; Figure 3 1 ). These residues are sufficiently far awa y from the active site that disturbance of the pK a of the proton donor/acceptor during catalysis is improbable.

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40 3.2.2 Engineering of Disulfide Bridges Disulfide bridges contribute to the enthalpic energy of the folded state with a covalent bond that has a typical bond dissociation energy of 60 kcal mol 1 ( 167 ) but there is also an entropic effect by decreasing the entropy of the unfolded state ( 168 ) This entrop ic effect of the disulfide bridge increases in proportion to the logarithm of the number of residues separating the two cysteines bridged. HCA IV is one of the most stable human isoforms known, with an exceptional resistance to solubilization in 5% SDS ( 169 170 ) The X ray crystal structure of HCA IV revealed that the presence of two disulfide linkages between res idues 6 11G and 23 203 may contribute to its stability ( 171 ) Incorporation of the disulfide linkage between residues 23 203 in HCA II enhanced the chemical stability in GuHCl from 0.9 M to 1.7 M resistance ( 172 ) Because chemical and thermal resistance to denaturation can be uncorrelated ( 173 174 ) this variant as well as the 6 11G disulfide alone and in conjunction with the 23 203 disulfide will be studied ( Figure 3 2). 3.2.3 Incorporation of Aromatic Ring Clusters Aromatic aromatic interactions (aromatic pairs ) are defined by a distance of less than 7.0 between the phenyl ring centroids. The following characteristics of aromatic pairs has been assigned: the interacting rings are close to perpendicular; most are involved in a cluster network; most link distinc t secondary structural elements; most are energetically favorable (potential energies between 0 and 2 kcal mol 1 ); and most take place between buried or partially buried residues ( 175 ) There are several examples in the literature of thermophilic proteins that contain these aromatic ring clusters ( 176 179 ) along with HCA IV ( 171 ) the only human isoform whose structure has been solved to contain such a n unique cluster. Mutation in HCA II to mimic this aromatic cluster in HCA

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41 IV will be performed via the following variants: L47 F, V49F, I146F and L212F ( Figure 3 3 ). 3.2.4 Introduction of Proline Residues I t has been proposed that proteins of known three dimensional structure can be stabilized by decreasing their entropy of unfolding ( 180 ) Proline, which can adopt only a few configurations and restricts the conformations of the preceding residues ( 181 ) has the lowest conformational entropy of all the amino acids. Thus, mutation s of Xaa Pro assuming the engineered residue does not introduce unfavorable strains in the protein 60 wh can adopt two groupings near 45 and +135 in the helical and sheet regions of the Ramachandran plot. These dihedral restrictrictions are due to the unique cyclic structure that proline adapts in that the C and amino hydrogens of the res idue preceeding proline is sterically restricted by the C bound to the imide nitrogen. There are a number of engineered thermophilic enzymes that have utilized this technique in the past with success ( 181 184 ) Residues Lys170, Glu234 and Leu240 in HCA II ( Figure 3 4) are the proposed proline mutation sites because they are located in rally adopt the required dihedral angles seen in the i turn ( 185 ) 3.2.5 Truncation of Flexible Surface Loops A current working hypothesis is that hyperthermophilic enzymes are more rigid than their mesophilic homologues. This is supported by numerous experimental data that includes frequency domain fluorometry and anisotropy decay ( 186 ) hydrogen deuterium exchange ( 187 189 ) and tryptophan phosphorescence ( 190 ) The belief is

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42 based off the observation that loops are usually the regions with the highest thermal factors in a protein crystal and, as such, are likely to unfold first during thermal denaturation. Loop truncation on several mesophilic proteins has increased the melting temperature in previous studies ( 167 191 ) and will be extended to include HCA II. The extended loop region at residue s 230 240 is a good candidate for truncation ( Figure 3 5 ; orange segment ) as it 1) has a higher average C B factor (thermal fluctuation) than the rest of enzyme 2) is not involved in any intramolecular hydrogen bond networks with the rest of the protein and 3) has tw o highly hydrophobic residues (Phe 231 and V al 240) that are solvent exposed. Furthermore, the carbonyl group of L eu 229 is sufficiently close to the amine group of M et 241 (2.8 ) that it can participate in a peptide bond without signifi cant backbone alteration ( Figure 3 5; green segment ). 3.3 Combination of Stabilizing Elements This study will comb ine the variants from Section 3.2 that displayed the greatest enhancement in stability without loss in catalytic efficiency into a single thermophilic variant of HCA II. To our knowledge, this will be the first attempt at incorporating multiple thermal sta bilizing elements into a mesophilic enzyme as most stabilization studies focus on one or two particular elements ( 167 ) These variants would be an attractive cand idate for biomedical and industrial applications (See Chap ter 2) The subsequent chapters will summarize the results from these experiments.

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43 Figure 3 1. Reduction of surface hydrophobicity on HCA II. The proposed nonpolar Leu triad (magenta) mutation s ites are shown in a surface representation of the enzyme.

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44 Figure 3 2. Proposed sites for the engineering of disulfide bridges in HCA II Mutation sites are highlighted in cyan

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45 Figure 3 3. Proposed mutation sites for the creation of an aromatic cl uster in HCA II Mutation sites are highlighted in green

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46 Figure 3 4. Proposed proline substitution sites on HCA II Mutation sites are highlighted in orange

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47 Figure 3 5. Proposed loop truncation site in HCA II Site is highlighted in orange wit h the potential peptide bond formation between residues 229 and 241 shown in green

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48 CHAPTER 4 REDUCTION OF SURFACE HYDROPHOBICITY 4.1 Overview To address the biomedical and industrial need for stabilized CAs while retaining desirable catalytic properties, five variants of HCA II were constructed. The three residues changed to investigate stability were selected out of ten measured using a random mutagenesis approach as described in US patent no. 7521217. These three residues were selected because they are l ocated on the surface of HCA II in a hydrophobic triad ( Figure 3 1) and were unlikely to adversely affect the catalytic rates These changes ser ved as the background to which the active site mutations Y7F ( 32 ) and N67Q ( 34 ) were added to create active HCA II variants with impr oved thermal stability and proton transfer rates To better understand the biophysical effect of thermal stabilizing mutations, X ray structures of the mutants were solved and enzyme kinetics were determined under a variety of possible industrial environme ntal conditions. These data show that changing hydrophobic surface residues in HCA II to polar moieties can improve stability through the introduction of intra and intermolecular forces Simultaneously it is possible to fine tune some of the enzyme kineti c parameters while creating variants with improved thermal stability. 4.2 Methodology 4.2.1 Protein Expression and Purification Site specific mutations of HCA II were made by GenScript. The first mutant, TS1 was constructed based on results reported in US patent no. 7521217 (filed by CO 2 Solutions) and contained the following single ami no acid substitutions: L100H as well as L224S and L240P. This triple mutant then served as the background for TS2 TS5. In

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49 addition to the starting triple mutations, TS2 a lso contained Y7F, TS3 had Y7F + N62L, TS4 had Y7F + N67Q, and TS5 had 6 mutations with Y7F + N62L + N67Q added. The corresponding cDNA for each variant was transformed in Escherichia coli BL21(DE3) cells in 1L of 2 x Luria broth medium containing ~0.1 mg/ mL ampicillin and grown at 37 C to a turbidity of ~0.6 at 600 nm. Protein production was induced with the addition of D 1 thiogalactopyranoside (IPTG) and ~1 mM zinc sulfate (final concentrations). The cells were incubated for an additional three hours and harves ted by centrifugation. A suspension of cells in 200 mM sodium sulfate, 100 mM Tris HCl, pH 9.0 was lysed by addition of hen egg white lysozyme and DNaseI with subsequent removal of cellular debris by centrifugation. The HCA II variants were purified on aff inity column containing an agarose resin coupled with p (aminomethyl) benzene sulfonamide, a tight binding inhibitor of HCA II ( 192 ) The bound HCA II was eluted with 400 mM sodium azide, 100 mM Tris, pH 7.0 followed by extensive dialysis in 50 mM Tris HCl, pH 7.8 to remove the azide. After purification the proteins were concentrated using Amicon Ultra concentration devices with a 10 kDa molecular weight cut off. Proteins were concentrated to 35 50 mg/mL prior to all subsequent experiments and characterizations. 4.2.2 X ray Crystallography The HCA II variants did not readily crystallize with the usual published conditions of ammonium sulfate or sodium citrate for HCA I I ( 32 ) and was screened in various crystallization conditions using the Gryphon robotic drop setter (Art Robbins Instruments ). The drops were set up using the vapor diffusion method against different commercial screens in 1:1 and 2:3 ratios o f protein to precipitant solution (drop sizes of

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50 1.0 and 0.8 L, respectively ) against 60 Diffraction quality crystals were obtained within a week from Hampton Screen 1, condition #6 ( 0.2 M magnesium chloride heptahydrate, 0.1 M T r is p H 8.5, and 30% w/v P EG 4000) in the 1:1 drops using a sample concentration of 50 mg/mL X ray diffraction data at 100K was collected on an RAXIS IV ++ using an in house rotating Cu anode HU H3R. Frames were collected with 1 oscillation steps and 5 minu tes per exposure. The crystal to detector distance was 80 mm. All crystals diffracted to between 1.56 and 2.0 resolution. The HKL2000 software package ( 193 ) was used to integrate, merge, and scale the data in the orthorombic space group P2 1 2 1 2 1 A summary of the data statistics is provided in Table 4 1. Initial phases for the varian ts were calculated using the coordinates of the high resolution, isomorphous HCA II structure (PDB: 3KS3; ( 68 ) ). The PHENIX.REFINE suite of programs ( 194 ) was used in cycles of restrained refinement of the molecular model, alternating with manual building using COOT ( 195 ) Table 4 1 shows a summary of the data set and refinement statistics. Experimental data and structural coordinates have been deposited with the Protein Da ta Bank and have the following accession numbers: TS1 = 3V3F, TS2 = 3V3G, TS3 = 3V3H, TS4 = 3V3I, TS5 = 3V3J. 4.2.3 Differential Scanning Calorimetry The DSC experiments were performed to assess the thermal stability of the HCA II variant s under near physi ological and acidic conditions using a VP DSC calorimeter (Microcal, Inc., North Hampton, MA) with a cell volume of ~0.5 mL. HCA II samples (6 10 M) were extensively buffer exchanged into 50 mM Tris HCl, pH 7.8. The samples and buffers were degassed, while stirring, at 16 C for 20 minutes prior to data collection. The DSC scans were collected from 20 to 100 C with a scan rate of 60 C/h.

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51 The calo rimetric enthalpies of unfolding were calculated by integrating the area under the peaks in the thermograms after adjusting the pre and post translation baselines. The thermograms were fit to a two state reversible unfolding model to obtain van't Hoff ent vH ) of unfolding. The HCA II variant melting temperature (T M ) values were obtained from the midpoints of the DSC curves, indicating a two state transition. The difference in Gibbs ( 196 ) : M (1 T/T M P ((T T M ) T ln (T/T M )) (4 1 ) M is the calorimetric enthalpy at T M P is the observed change in heat capacity between the folded and unfolded states. The denaturation enthalp ies law equations ( 68 ) : M P (T T M ) (4 2 ) M P ln (T T M ) ( 4 3 ) Thermograms for all samples were obtained in triplicate and then averaged to obtain the final profile, which was then used for reference subtraction and data analysis. 4.2.4 Kinetic Studies The 18 O exchange method i s based on mass spectrometric measurements using a membrane inlet of the depletion 18 O from CO 2 ( 197 ) The isotopic content of CO 2 in

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52 solution is measured when it passes across a membrane and into an Extrel EXM 200 mass spectrometer. The measured variable is the atom fraction of 18 O in CO 2 Th e first step of catalysis has a probability of reversibly labeling the ZnOH with 18 O (Eq. 4 4 ). During the next step the 18 OH can be protonated and results in the release of H 2 18 O to the bulk solvent where it is essentially infinitely diluted by H 2 16 O (E q. 4 5 ). In this process, His64 acts as a proton shuttle (Tu et al., 1989). HCOO 18 O + EZnH 2 O EZnHCOO 18 O COO + EZn 18 OH (4 4 ) H + His64 EZn 18 OH + H 2 O His64 EZnH 2 18 O His64 EZnH 2 O + H 2 18 O (4 5 ) The 18 O exchange method obtains two different rates at chemical equilibrium ( 197 ) : R 1 wh ich is the rate of exchange of CO 2 and HCO 3 (Eq. 4 6 ); and R H2O which is the rate of release of H 2 18 O from the enzyme (Eq. 4 7). In Eq. 4 6 k cat ex is the rate constant for maximal conversion between substrate and product while K eff S is the effective bin ding constant of substrate ([S] is the concentration); [S] can be either CO 2 or HCO 3 depending on the direction of the rea ction. The ratio expressed in Eq. 4 6 of k cat ex /K eff S is in principle the same as k cat /K M obtained under steady state conditions. R 1 /[E] = k cat ex [S]/(K eff S + [S]) ( 4 6 ) In the second step of catalysis the rate R H2O is the part of 18 O exchange that is dependent on the rate of proton transfer from His64 to the labeled Zn OH (i.e. in the dehyd ration direction) ( 23 ) Eq.4 7 shows the relationship between k B the rate constant

PAGE 53

53 for proton transfer to ZnOH and (K a ) donor and (K a ) ZnH2O that are the ionization constants for the proton donor and Zn H 2 O, respectively. R H2O = k B /[[1 + (K a ) donor /[H + ]] [1 + [H + ]/(K a )/ ZnH2O ]] ( 4 7 ) Except f or the temperature dependence studies, all enzyme kinetic measurements were done at 25 C in the absence of buffer using a total substrate concentration (all species of CO 2 ) of 25 mM. The temperature dependence studies used 10 mM total species of CO 2 Kine tic constants and ionization constants shown in Eqs. 4 6 and 4 7 were determined through nonlinear least squares methods (Enzfitter, Biosoft). Enzyme activity as R 1 /[E] was also measured at temperatures from 10 C to 70 C in the similar solutions (100 mM HEPES and 10 mM total substrate) at pH 7.6. After the reaction solution was equilibrated to each different temperature, a small sample of enzyme (at 0.1 0.2% of reaction volume) was added. Measurements of 18 O content of CO 2 were made over the following o ne to five minutes. These are rough estimates of the thermal inactivation temperature with measurements made in intervals of 5 C at the higher temperatures the purpose of which was to determine whether there was inactivation of catalysis at a temperature less than the major unfolding determined by DSC. 4.3 Results & Discussion 4.3.1 X ray Crystallography All variants crystallized in the orthorhombic P2 1 2 1 2 1 space group and were highly isomorphous with approximate unit cell dimensions a = 42, b = 72, c = 7 5 and diffracted to between 1.6 and 2.0 resolution. All models refined to R cryst and R free

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54 between approximately 16 19% and 20 25%, respectively. Table 4 1 shows a summary of all data set and refinement statistics. Leu100, Leu224, and Leu240 are a ll between 8 14 from each other (C C distance) and form a small hydrophobic patch on t he surface of HCA II (Figure 4 1A ). This patch is at least 20 away from the enzyme active site. The mutation sites are located within the interface of two cryst allographic symmetry related chains which may contribute to different c rystal packing compared to wild type HCA II. Additionally, the decrease in surface hydrophobicity likely contributes to the increased solubility and different crystallization conditions required to crystallize the variants Based on a comparison of the TS1 (PDB: 3V3F) and wild type (PDB: 3KS3) X ray crystal structures it is clear that changing these hydrophobic residues to polar residues results in increases stability through differences in enthalpic contributions with the gain of hydrogen bonds and favorable electrostatics ( Figure 4 1 ,B D ). L100H accommodates weak hydrogen bonds (3.0 3.4 ) with the backbone amide of Gly102 and the side chain of Gln103 ( Figure 4 1B ). The average B fac tors of all atoms in the loop consisting of residue 97 to 104 is ~15 2 and are similar compared to wild type structures also determined at 100K. This supports that an increase in enthalpic contributions (hydrogen bonds) at position 100, and not entropic ( thermal movement ) dominates the observed increase in stability. L224S is observed making hydrogen bond interactions to the backbone carbonyl of Glu221, where as a water molecule coordinates to the gaunidino group of Arg227 and the carbonyl group of Ser220 ( Figure 4 1C ). A comparison of the average B factors for all atoms from residue 223 225 is interesting: for wild type it is ~12 2 and 19 and 26

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55 2 for TS2 and TS 4, respectively. This implies an increased thermal fluctuation in the mutant compared to wi ld type that is also reflected by the disorder of Ser224 in TS2 L240P creates a solvent accessible, hydrophili c pocket that allows for the ordering of two water molecules to make hydrogen bonds with the sidechain of Asn230 to the carbonyl group of Arg227 ( Figure 4 1D) The introduction of a P ro at position 240 which sits at the end of a surface loop, may be expected to cause a reduction in the loop flexibility. However, similar to the mutations introduced at positions 100 and 224, changing L240P appears to increase the aver age B factor of the loop from ~ 12 to 35 2 As expected, there are no significant active site differences between wild type ( Figure 4 2A) and TS1 ( Figure 4 2B) when superimposed onto one another. The positions of the waters involved in proton transfer and the configurations of the active site re sidues are comparable ( 25 26 ) In TS2 ( Figure 4 2C) wh ich contains the Y7F mutation, W3A i s displaced as compared to wild type HCA II resulting in a more linear proton transfer network, consistent with previously published resul ts ( 32 ) The remaind ing waters and r esidues in the active site are comparable to wild type HCA II. In TS3 (containing Y7F/N62L; Figure 4 2D) the solvent W3B has moved but is still interacting with Asn67 and W2. Similarly to TS2, the introduction of a hydrophobic residue causes displacement of active site water molecules. In TS4 (containing Y7F/N67Q; Figure 4 2E) Gln67 m aintains a similar binding pattern t o W3B as seen in wild type HCA II In TS5 (containing Y7F/N62L/N67Q; Figure 4 2F), the proto n transfer water network has been altered in that W2 makes direct hydrogen bonds with His64 and a water molecule pushed towards Asn244 as well as a weak potential hydrogen bond with Gln67 Interestingly, W3B is still seen to interact with Gln67, but no lon ger to W2.

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56 These results are consistent with previous structure function studies of HCA II ( 32 34 ) and indicates that the presence of the surface Leu mutations do no t affect the active site structure in the TS mutants. 4.3.2 Stability To test the stability of the variants against denaturation through thermal means, a series of experiments was carried out by DSC ( Figure 4 3) and by measuring the rate of catalyzed CO 2 / HCO 3 interconversion. DSC scans were measured for wild type, Y7F mutant and each of the TS HCA II variants. The melting temperatures or major unfolding transitions (T M ) for each of the variants occurred at distinct peaks in the thermograms. The average pe ak values (with standard deviations shown in parentheses) from triplicate runs are given in Table 4 2 Wild type HCA II had a T M of ~58 C ( Figure 4 3A) under these experimental conditions and introducing the three backgrou nd mutations present in TS1 (L10 0H L224S, L240P ) increased the T M to 65 C ( Figure 4 3B; Table 4 2 ). Introducing the Y7F mutation to increase proto n transfer rate in TS2 had a destabilizing effect ( Figure 4 3C) reducing the T M to 53 C while adding the background mutations restores the stability to ~61 C Addition of N67Q to an active site containing Y7F stabilizes HCA II almost back to wi ld type levels while also displaying high catalytic efficiency ( Table 4 2) As such, TS4 HCA II displayed an intermediate thermal resistance ( Figure 4 3E), inbetween TS1 and TS2. The T S3 and TS5 (Figs. 4 3D and F, respectively ) variants, both containing the N62L mutation, were seen to have a decrease in thermal stabilization relative to the other TS variants and comparable to wild type levels. Among a ll of the variants of Table 4 2 TS1, TS2, and TS4 have values of T M that are significantly higher than wild type HCA II. This is very encouraging as these variants

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57 have not only different stabilities but also somewhat diffe rent ki netic profiles ( Figure 4 4 ) compared to wild type that make them interesting from an i ndustrial point of view 4.3.3 Catalytic Activity The pH profiles were determined for R 1 the rate of catalyzed interconversion of CO 2 and bicarbonate (E q 4 6 ), and R H2O the rate of dissociati on of H 2 18 O from the active site ( Eq. 4 7 ; Figure 4 4A ) ( 93 ) The background mutations (L100H, L224S, L240P ) did not significantly affect the rate of CO 2 hydration r eflected through k cat ex /K eff S (Table 4 2 ) or R 1 /[E] ( Figure 4 3B) Moreover, the replacements at positions 7, 62, and 67 also caused no significant changes in these measures of th e first stage of catalysis (Eq. 4 4 ). This result was expected since the surfa ce Leu mutations and the amino acid replacements in TS 1 5 are sufficiently far from the catalytic Zn to avoid structural and electrostatic disrupti ons of the reaction of Zn OH with substrate. T here are interesting differences however, in the rate consta nts R H2O /[E] ( Figure 4 4A ) and the rate constant for proton transfer in catalysis k B (Table 4 2 ). The rate constant k B measures in large part the proton transfer along an ordered water structure between His64 and the Zn OH in the dehydration direction and is determined from the bell shaped pH profiles such as observed w ith wild type HCA II ( Figure 4 4A ) ( 23 40 197 ) These results show that the background replacements of surface Leu residues in TS1 do not negatively affect R 1 or k B co mpared with wild type (Table 4 2 ), consistent with their location far from the active site and proton transfer pathw ay ( 40 ) However, combining the background mutations in TS1 with specific active site changes at position 7 and 67 caused an unexpe cted, albeit modest, boost in proton t ransfer activity ( Table 4 2 ) consistent with other data ( 34 )

PAGE 58

58 4.4 Discus sion Altered loop conformations between residues Val37 S er50, Phe70 Lys80 and Lys225 Pro 240 are observed in these mutants relative to the wild type structure ( 93 ) The C backbone trace of the Phe70 Lys80 loop is displaced by up to 3.3 compared to wild type with the most dramatic effect arising from the movement of Lys76 that now makes a hydrogen bond w ith Asp71 with Gln74 These alternative loop conformations have been observed with HCA II:inhibitor structures solved in the orthorhombic space group as well as for other HCA II structures with mutations in or near the opening of the active site ( 198 199 ) These displaced surface loops are most likely a direct consequence of crystal packing forces. As a result of the crystallization conditions used for the TS mutants of HCA II, a single Cl is seen along the surface within H bonding distance of the amide groups of Gln158 and Lys225 for TS1 4. In the TS5 structure Lys225 is not in position to interact with a Cl which allows Glu158 to be present at the Cl binding site. As a result, a water molecule is observed at this position instead. It is not obvious why Lys225 occupies this unique conformation in TS5 compared to TS1 4. Additionally, previous studies ( 199 ) of HCA II in an orth orhombic space group report a zinc metal coordinated to His4 located near the opening o f the active site cavity ( PDB: 2X7S). Interestingly TS2 (PDB: 3V3G) displays a similar density in the 2F o F c map in this re gion as well group of residue His3 is coordinating to the observed density along with possible interactions from His64, the symmetry related His36 residue, and a water molecule. Attempts at placing a Zn 2+ ion in this density resulted in increased B fa ctors (> 80 2 ) in addition to appearance of negative density in the F o F c map. Due to the uncertainty at this position, a water molecule was built and refined (B factor < 20 2 ). Nevertheless, it

PAGE 59

59 is worth noting that the possible Zn coordination site at t he N terminus and crystallographic contact point is very similar to the canonical Zn His arrangement found in the active site. The chemical stabilities of the mutants were compared to wild type HCA II increasing urea concentration up to 8 M in 1 M incremen ts while measuring the kinetic constant R 1 /[E] ( 93 ) The Leu surface mutations had no significant effect on enzyme ability to withstand denaturation by urea under th ese conditions. The addition of 4 M urea led to less than 10% relative activity compared to no urea f or all variants, including wild type. This observation may be explained by urea denaturation through unfolding of the hydrophobic core of the protein inclu ding an indirect effect on water structure and the integrity of hydrophobic interactions that form the interior of the enzyme. Since the variants reported here were all surfac e and active site of HCA II and not in the core, the denaturation resulted in si m ilar effect in the wild type and variant enzymes. Not dissimilar to the chemical stability, the thermal unfolding transitions described above varied for the TS HCA II variants Studies of protein unfolding find differences in thermal and chemical stabilit y examined using experimental and computational methods ( 174 ) From a structural perspective the rationale for how these mutations confer stability is not intuitive. In contrast to the B FIT approach, an increase in the thermal fluctuation of residues at positions 224 and 240 is observed with a concomitant increase in thermal stability ( 150 ) This probably reflects the dominant effect of the gain in hydroge n bonding and hydrophilicity over flexibility on the surface of HCA II. The underlying principle of thermal stability as a change in surface electrostatics reported

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60 here for HCA II is consistent with several other successful studies on diverse enzymes such as acylphosphatase, lactalbumin and ubiquitin ( 144 146 148 ) Incorporation of mutations in the active site of HCA II resulted in loss of intermolecular hydrogen bond interactions for the variants TS3 and TS5 between active site residues and water molecules ( Figure 4 2C and E). Loss of hydrogen bonds to t hese water molecules in the active site at residues 7 and 62 seem to have a significant effect on the T M of these variants compared to the other TS active site mutations (Table 4 2). This is consistent with a decrease in T M seen in the Y7F variant compared to wild type HCA II (Table 4 2). The notion of loss of hydrogen bonds to water molecules would destabilize the enzyme correlates well to the observed stabilization seen upon introduction of water molecules with the mutations L224S and L240P ( Figure 4 1C a nd D). T he proton transfer efficiency (k B ) of the TS variants can be understood in terms of the results for the corresponding variants with single amino acid replacements. The value of k B for Y7F HCA II is increased about 5 fold co mpared with wild type (Ta ble 4 2) and is comparable to previous studies ( 32 ) T he values of k B for the variants containing the Y7F mutation (TS2 and TS4) are comparable at 5.6 and 4.9 s 1 respectively. The value of k B for N67Q HCA II is increased about 2 fold co mpared with wild type ( 34 ) and for N6 2L HCA II the value of k B is decreased 8 fold. These factors then influence the values of k B shown in Table 4 2 ; for example, the two var iants (TS3 and TS5) containing N62L have low values of R H2O /[E]. The values of k B measuring proton transfer come into s ignificance when HCA II is under maximal velocity conditions for concentrations of CO 2 well above 10 mM. The rate constant R1/[E] was determined while increasing

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61 temperatur It is seen that the TS1, TS2, and TS4 variants maintain their ca talytic activity at 65 C, whereas the other variants of this figure show considerable decrease ( Figure 4 4B ) 4.6 Conclusions This study has shown that it is possible to enhance the thermal stability of HCA II by strategic replacements of amino acids on t he surface of the enzyme. Moreover, these replacements had no significant effect on the active site structure and no effect on the catalytic rate of CO 2 hydration and HCO 3 dehydration. Single amino acid replacements that were previously found to enhance c atalysis were also effective in enhancing catalysis in variants with these surface changes. The net result was a variant of HCA II (TS2 and TS4 of Table 4 2 ) with thermal stability enhanced by approximately 6 C and with maximal proton transfer enhanced ap proxim ately 6 fold compared with wild type HCA II. The initial results reported here shed light on the underlying biophysical principle, i.e. removing surface hydrophobic residues and replacing them with polar or hydrophilic residues leads to a gain in hyd rogen bonding interaction and these results in increased thermal stability. Other hydrophobic residues on the surface of HCA II that are potential mutations sites include: Leu44, Leu57, Leu60, Val78, Leu84, Val109, Val163, Leu185, Leu189, Val223, Leu229, P he231, Val242, Leu251 and Ile256.

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62 Table 4 1. X ray crystallographic data set and refinement statistics for the TS HCA II variants TS1 TS2 TS3 TS4 TS5 PDB Accession Number 3V3F 3V3G 3V3H 3V3I 3V3J Resolution () 20 2.0 (2.07 2.0 0 ) 20 1.56 (1. 60 1.56) 20 1.85 (1.95 1.85) 20 1.74 (1.80 1.74) 20 1.63 (1.69 1.63) Total number of measured reflections 96743 227265 101034 110298 178801 Total number of unique reflections 14737 32937 19063 20425 28381 R sym a (%) 12.4 (36.7) 7.8 (48.8) 9. 6 (47.7) 9.6 (33.5) 7.0 (48.9) 7.6 (3.5) 19.9 (3.3) 13.2 (3.1) 7.2 (2.9) 18.8 (3.3) Completeness (%) 92.9 (90.2) 99.4 (97.9) 95.5 (93.7) 82.7 (99.4) 99.3 (97.6) Redundancy 6.7 (6.7) 6.9 (6.6) 5.3 (5.3) 3.0 (3.1) 6.3 (5.6) R cryst b (%) 18.8 17.0 16.5 19.5 18.3 R free c (%) 25.6 20.0 22.4 25.3 21.9 Residue Nos. 257 258 258 257 258 No. of Protein atoms (including alternate conformations) 4031 4112 3994 3942 4001 No. of water molecules 142 277 220 89 195 R.m.s.d: Bond lengths (), angles () 0.012, 1.3 7 0.009, 1.25 0.010, 1.26 0 .011, 1.31 0.009, 1.29 Ramachandran statistics (%): Most favored, additionally allowed, and generously allowed regions 96.1, 3.9, 0.0 97.0, 3.0, 0.0 96.9, 3.1, 0.0 97.3, 2.7, 0.0 96.5, 3.5, 0.0 Average B factors () 2 : All, Main side chain, solvent 24.9 22.5, 26.9, 25.5 21.0, 17.5, 22.5, 27.0 21.6, 19.3, 22.9, 24.6 24.2, 21.1, 26.9, 22.9 16.8, 14.2, 18.2, 21.7 r.m.s.d. C () d 0.45 0.45 0.47 0.47 0.53 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensities. b R cryst o F c o |) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refi nement (5% of reflections for all data sets). Values in parenthesis are for the highest resolution shell.

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63 Table 4 2 Thermal stability and catalytic m easurements of the TS HCA II variants Enzyme T M ( C) a k cat ex /K eff S ( M 1 s 1 ) b k B 1 ) b wild t ype HCA II 58 ( 1 ) 120 0.8 Y7F 53 ( 1) 120 c 3.9 c Y7F + N67Q 56.9 ( 0.3) 75 d 12 d TS1 (L100H, L224S, L240P) 65 ( 2) 85 1.3 TS2 (TS1 + Y7F) 61.1 ( 0.5) 110 5.6 TS3 (TS1 + Y7F + N62L) 58.1 ( 0.3) 88 ~ 0.1 TS4 (TS1 + Y7F + N67Q) 62.7 ( 0.5) 94 4.9 TS5 (TS1 + Y7F + N62L + N67Q) 59.2 ( 0.4) 110 ~ 0.1 a Denaturation temperature as determined by DSC. b The standard errors in these rate c onstants determined by fitting Eqs. (4 6) and ( 4 7) to the data of Figures S2 and S3 are in the range of 10% to 20%. c Data from Fisher et. al. ( 32 ) d Data from Milkulski et. al. ( 34 )

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64 Figure 4 1 X ray crystallographic model of the TS HCA II variants A ) HCA II in blue with the mutated Leu residues in orange the zinc is shown as a magenta sphere with the hydrophilic residues inv olved with catalysis shown in stick. For panels (B D) wild type and TS1 (PDB: 3V3F) was superimposed to show the differenc es, wild type is in blue and TS1 is in cyan stick B ) L100H variant and inferred hydrogen bonds (based on distance and angles) ar e sho wn as black dashed lines, C) L224S, D ) L240P and waters and hydrogen bonds.

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65 Figure 4 2. Active site comparison of stabilized HCA II variants. A) Wild type HCA II active site (PDB: 3KS3). The Zn 2+ metal is shown as a magenta sphere and all the hydrophil ic active site residues (and their counterparts in the mutants) are shown as sticks, waters are shown as red spheres. Residues and waters are as labeled and inferred hydrogen bonds are shown as black dashed lines. The stabilized variant active sites are sh own for B) TS1 C) TS2 D) TS3 E) TS4 and F) TS5.

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66 Figure 4 3. Thermograms for TS HCA II v ariants. Black line shows experimental data whereas the red line is the fit to the data. A) wild type HCA II B) TS1 C) TS2 D) TS3 E) TS4 and F) TS5.

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67 Figure 4 4 Catalytic Activities of TS HCA II v ariants. A) Profile of R H2O /[E] (s 1 ) as a function of pH for wild type and variants of HCA II. Wild type = black square ; TS1= red diamond ; TS2 = blue triangle ; TS3 = brown square ; TS4 = green circle; TS5 = magenta tria ngle B) The rate constant R 1 /[E] (s 1 ) as a function of temperature for the interconversion of CO 2 and bicarbonate catalyzed by variants of HCA II Data have been separated on the ordinate to avoid superposition of points. Solutions contained 10 mM of all species of CO 2 and 100 mM HEPES at pH 7.6.

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68 CHAPTER 5 ENGINEERING OF DISULFIDE BRIDGES 5.1 Overview A conserved disulfide bridge has been observed between residues 23 and 203 (HCA II numbering) for all of the membrane bound human isozymes of CA (HCA IV, I X, XII and XIV) as well as in the secreted isoform HCA VI ( 200 ) The se disulfide bridges are thought to confer extra stability experienced in the harsh extracellular environment in which these isoforms are exposed and could provide an explanation for the resistance to denaturation by SDS seen in HCA IV ( 169 ) Additionally, a CA isolated from Neisseria gonorrhoeae also contains a disulfide bridge at the aforementioned posit ion ( 201 ) and was shown to remain globular up to 2.1M concentration of guanidine hydrochloride (GuHCl) ( 202 ) The disulfide linkage is also conserved in the X ray crystallographic structure of a hyper thermophilic CA isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 found in a hot spring in Yellowstone National Park and could provide a source for the enzymes remarkable stability (T M > 100 C) ( 203 ) HCA IV also has a unique disulfide linkage between residues 6 11G (11G being the seventh residue in a loop insertion in the HCA IV amino acid sequence relative to HCA II). Given the need for a thermal stable varia nt of CA that can withstand the high temperatures and acidic environment of current carbon sequestration protocols, previous studies ( 172 ) that engineer a disu lfide lin kage between residues 23 and 203 (A23C/L203C ) into C206S HCA II (HCA II C206S ) has been extended Thi s disulfide containing variant (DS1) was previously shown ( 172 ) from tryptophan fluorescence measurements to have an approximate 2 fold increased resistance to GuHCl induced denaturation compared to HCA II C206S (1.7M versus 0.9M, respectively), corresponding

PAGE 69

69 to a net stabilization of 6.6 kcal mol 1 Howev er, the relationship between chemical and thermal denaturation has been shown to be uncorrelated in some proteins ( 174 ) Additionally, the secondary disulfide linkag e found in HCA IV (residues 6 and 11G) has been incorporated in conjugation with the DS1 mutations (A23C/L203C/C206S) to create the DS2 HCA II variant (G6C/N11C/A23C/L203C/C206S). I n this study DSC was used to assess the thermal stability of DS1 and DS2. It was revealed that these variant s have significantly enhanced thermal tolerance at near physiological conditions as well as in an acidic environment compared to that of wild type HCA II. Moreover, depletion of 18 O label from CO 2 measured by mass spectrom etry revealed comparable catalytic efficiency of DS1 to wild type HCA II but with a higher tolerance for elevated temperatures. Unfortunately, the catalytic efficiency of DS2 was decreased two fold compared to HCA II. Lastly the X ray crystal structure of DS1 HCA II was solved to 1.77 resolution and showed the successful formation of a disulfide linkage in HCA II The DS2 HCA II variant has resisted all crystal formation attempts thus far. 5.2 Methodology 5.2.1 Enzyme Expression and Purification HCA II c DNA containing the DS1 (A23C/L203C/C206S) and the DS2 (G6C/N11C/A23C/L203C/C206S) mutations was prepared from an expression vector containing the enzyme coding region ( 141 ) via site directed mutagenesis using the Stratagene QuikChange II kit and primers from Invitrogen. The variant cDNA was transformed into Escherichia coli XL1 Blue super competent cells, which were then confirmed by DNA sequencing of the entire cod ing region. Enzyme expression and purification was carried out as detailed in Section 4.2.1 with the addition that o xidized

PAGE 70

70 glu tathione was added to the purified sample to a final concentration of ~0.1 mM to induce disulfide formation ( 172 ) The oxidized sample was then concentrated to ~10 mg/mL via centrifugal ultra filtration using a 10 kDa molecular weight cutoff filter (Amicon). Possible intermolecular disul fide dimeric complexes were then removed via size exclusion chromatography on a Superdex 75 column using the dialysis buffer and a flow rate of 0.5 mL/min. The absence of dimeric DS1 complexes were confirmed via visual inspection of native gel electrophore sis (data not shown) 5.2.2 Crystallization and Diffraction Data Collection Crystals of the DS1 HCA II variant were observed after one week in a 1:1 mixture of protein: reservoir solution using the hanging drop (5 L) vapor diffusion method against a reser voir (500 L) of 1.3 M sodium citrate, pH 9.0. Diffraction data were collected at 100 K on an in house Rigaku R Axis IV ++ image plate detector using an RU H3R rotating Cu anode (K = 1.5418 ) operating at 50 kV, 22 mA with a crystal to detector distance o f 80 mm. The X rays were focused via Osmic optics, followed by a helium purged beam path. Diffraction data were collected at 1 oscillations with an exposure time of 300 seconds. The HKL2000 software package ( 193 ) was used to integrate, merge, and scale the data in the monoclinic space group P2 1 to a resolution of 1.77 A summary of the dat a statistics is provided in Table 5 1. Initial phases for the DS1 HCA II variant were calculated using the coordinates of the high resolution, isomorphous wild type HCA II structure (PDB: 3KS3; ( 68 ) ). The PHENIX.REFINE suite of programs ( 194 ) was used in cycles of restrained refinement of the molecular model, alternating with ma nual building using COOT ( 195 ) The electron density map was weak for the disordered N te rminal residues 1 3, but the disulfide linkage at residues 23 and 203 as well as the pseudo wild type C206S mutation were

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71 evident in the initial F o F c difference map, with subsequent refinements showing excellent electron density in the 2F o F c maps at these positions. Solvent molecules refined with B factors of more than 50 2 were excluded from the final model. The R cryst and R free values for the final model were 15.0 and 18.9%, respectively. The PROCHECK algorithm ( 204 ) was u sed to assess the quality of the final model. The final model statistics are included in Table 5 1. All structural figures were generated in PyMOL ( 205 ) Experimental data and structural coordination have been deposited with the Protein Data Bank (http://www.rcsb.org) with the accession number 4HBA. 5.2.3 Differential Scanning Calorimetry DSC protocols for samples at pH 7.8 are as outlined in Section 4.2.3 Additionally, studies at pH 5.6 were conducted for wild type HCA II and the DS1 HCA II variant Sampl es (1 mg/mL) were buffered excha nged into 80 mM sodium citrate, 20 mM citric acid at pH 5.6. Thermograms were carried out in triplicate and analyzed as detailed in Section 4.2.3 5.2.4 Catalytic Activity Measurements of R 1 and R H2O were performed for all samples at 25 C in the absence o f buffer using a total substrate concentration (all possible isotope labeled species of CO 2 ) of 25 mM and an enzyme concentration of ~1 mg/mL. The enzymatic activity was also studied at a temperature range from 10 C to 70 C in 100 mM HEPES and 10 mM subs trate at pH 7.6 for DS1. After the reaction was equilibriated to each temperature, a small sample of enzyme (~0.2% v/v) was added to the reaction vessel. Measurements of 18 O content of CO 2 were made over the following one to five minutes. The kinetic const ants and ionization constants shown in Eqs. 4 6 and 4 7 (Section 4.2.4) were determined through nonlinear least squares methods (Enzfitter, Biosoft).

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72 5.3 Results 5.3.1 X ray Crystallographic structure of DS1 The DS1 HCA II variant crystallized in the monoc linic P2 1 space group with unit cell dimensions a = 42.3, b = 41.2 and c = 71.6 , 104.2 o and diffracted to a maximum resolution o f 1.77 . The final model ( Figure 5 1A ) refined to a R cryst and R free of 1 5.0 and 1 8.9 %, respectively. Table 5 1 shows a summary of the collected diffraction sets and final refinement statistics. T he disulfide linkage is well visualized in the final calculated 2F o F c map at an contour level of 1.4 ( 206 ) There is very little deviation in the overall structure of the DS1 HCA II variant when aligned with the C backbone of wild type HCA II (0.2; Table 5 1) but there are, however, some local perturbations around the mutation sites. Interestingly, the C atom of residue 23 in the DS1 variant is shifted ~0.8 compared to wild type HCA II, whereas the C at residu e 203 is the same in both s tructures ( Figure 5 1B ). This shift in the backbone around residue 23 is presumably a direct result of the disulfide formation. In addition, the relative identity in the backbone of residues proximal to residue 203 in DS1 compar ed to that of wild type HCA II is advantageous in that it does not distort the residues known to be important in the catalyti c activity of the enzyme ( Figure 5 1C ). As such, the crystal structure suggests that the kinetic activity of DS1 HCA II should corr eleate well with that of wild type HCA II as no significant variations are observed in either the backbone or the side chains of residues lining the catalyic site. 5.3.2 Thermal Stability of Engineered Disufide Linkages The thermal unfolding transitions o f the disulfide HCA II variatns and wild type HCA II were studied in triplicate utilizing DSC. A major unfolding transition, the observed dominant peak, was seen in all samples ( Figure 5 2). The transitions were calculated to

PAGE 73

73 be endothermic and were center ed at the T M with the maximum heat capacity (C P ) occurring at the midpoint of the peak. The results of DSC analysis are summarized in Table 5 2. The T M of DS1 HCA II at pH 7.8 was measured to be 71.0 0.1 C ( Figure 5 2A) revealing remarkable stability when compared to wild type HC A II which showed an unfolding transition at 57.1 0.1 C ( Figure 5 2B) ). This enhanced stability was conserved at the more acidic pH of 5.6 for which DS1 HCA II showed a T M of 59.6 0.1 C ( Figure 5 2C) whereas wild type HCA II showed less resistance to the acidic environment with a T M o f 45.4 0.1C ( Figure 5 2D ). The increased thermal and acid tolerance of the DS1 HCA II variant correlates well to its observed chemical stabili ty in high concentrations of Gu HCl when co mpared to HCA II C206S ( 172 ) The calorimetric M ) was calculated via integration of the area under the unfolding peak, normalized to the protein molar concentrations and revealed values of approximatel y 310 and 220 kcal mol 1 for DS1 and wild t ype HCA II at pH 7.8, respectively. This increased enthalpic contribution to the overall stability of the DS1 variant compared to wild type HCA M values of 200 a nd 150 kcal mol 1 respectively. The thermogram for the DS2 HCA II variant ( Figure 5 2E) at pH 7.8 revealed a similar denaturing profile as DS1 (three transition states) but each peak was destabilized by ~3 C. The T M for the dominant peak for the DS2 HCA II variant is ~68 C, whereas the less dominate peaks were present at ~53 and 63 C. The approximate 10 C increase in thermal stability of the DS2 HCA II variant containing two disulfide bridges compared to the native enzyme is lower than that observed with the addition of one disulfide linkage betwee n residues 23 and 203 in DS1 HCA II (Table 5 2). This

PAGE 74

74 could be due to the addition of a nonconserved linkage at the N terminus of DS2 HCA II imposing structural reconfigurations around the mutation sites. An X ray crystallographic structure of the DS2 HCA II variant is needed to elucidate the biophysical principles underlying this destabilization effect as compared to the DS1 HCA II variant. 5.3.3 Catalytic Activity The pH profiles for the two rate constants, k cat /K M and R H2O were determined by measuring the exchange of 18 O between CO 2 and water determined by mass spectrometry. The rate of catalyzed interconversion between CO 2 and bicarbonate as measured by k cat /K M (Eq. 4 6 ; Figure 5 3A; Table 5 2 ) for DS1 HCA II was similar to that previously reported for wild type HCA II ( 32 ) These results are expected as the disulfide location is more than 11 away from the catalytic ZnOH and no perturbations in the C backbone lining the active site are observed when overlayed with a HCA II crystal structure ( Figure 5 1C ). The catalytic efficiency of DS2 has been reduced by two fold compared to HCA II ( Figure 5 3B; Table 5 2). The value of k B the rate constant for pr oton transfer, from Eq. 4 7 indicates a slight reduction for the DS1 and DS2 HCA II variant s when compared to wild type HCA II ( Figure 5 3 C and D ; Table 5 2 ). This decrease in proton transfer has been measured before via spectrophotometric measurements in the DS1 HCA II variant ( 172 ) The inclusion of disulfide bridge between residues 6 and 11 in DS2 HCA II may reconfigure the N terimnus (and thus Tyr7) which is important in the positioning of the water molecules involved in the proton transfer network (see Figure 1 1B), resulting in lowered catalytic efficiency (Table 5 2). To assess the stability of the DS1 HCA II variant at higher temperatures, the rate consta nt of the dehydration of bicarbonate to CO 2 k cat /K M was measured in 5 C increments from 10 to 70 C ( Figure 5 3E ) giving an approximation of the thermal

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75 inactivation temperature. The thermal inactivation temperature, T inact was estimated as the data poi nt at which the rate constant k cat /K M for solution containing enzyme was found to decrease to a value close to the uncatalyzed reaction. Following these guidelines, HCA II showed a significant loss in catalytic activity at 60 C whereas the DS1 variant sho wed no loss of activity up to 70 C, the temperature limi tat ion of the experiment ( Figure 5 3E ; Table 5 2 ). These values also correlate well to the T M values measured by DSC in similar conditions (Table 5 2 at pH 7.8) and show that the thermal unfolding te mperature is also the thermal inactivation temperature for HCA II. 5.4 Discussion The overall geometry of the engineered disulfide bridge in HCA II at residues 23 and 203 correlates well to earlier prediction models ( 172 ) as well as in other X ray crystallographic models of other CAs where this disulfide linkage is conserved ( 2 06 ) The configuration of the disulfide bridge between residues 23 and 203 can be described as a (+) right handed hook, one of the most common conformations of disulfide bridges, when analyzing the gauche magnitudes of the angles that form the bridge ( 207 ) The dihedral strain energy (DSE) of the the disulfide can be calculated from the values of the five angles using the relationship ( 206 208 209 ) : DSE (kJ mol 1 ) = 8.37(1 + cos 3 1 ) + 8.37(1 + cos 3 1 ) + 4.18(1 + cos 3 2 ) + 4.18(1 + cos 3 2 ) + 14.64 (1 + cos 2 3 ) + 2.51(1 + cos 3 3 ) (5 1 ) Although Eq. 5 1 only accounts for values for the dihedral angles of a disulfide bridge and not other factors such as bon d lengths and van der Waals contacts, this relationship has been used to provide a semi quantitative analysis of the strain in a

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76 disulfide bond ( 210 213 ) Using the observed angles in the crystallographic model, the DSE of the disulfide in DS1 HCA II is calculated to be 21.5 kJ mol 1 This DSE value is well within the possible values of 2.0 84.5 kJ mol 1 allowed for disulfide bridges, and is comparable to the mean DSE for other right handed hook disulfide formations (20.8 kJ mol 1 ) found in the protein data bank ( 207 ) Comparison of the geometry for the engineered disulfide bond in DS1 HCA II to that of other X ray crystallographic models of CAs where this linkage is conserved show that the right handed ho ok conformation is retained, albeit with varying magnitudes of gauche values for 1 and 1 and DSE values ( 206 ) T he thermograms obtained from DSC for DS1 and wild type HCA II at acidic a nd near physiological pH ( Figure 5 2 ) were fit to a two stat e reversible unfolding model to 5 2). Most of the transition curves fit well to this model, showing a single, narrow endothermic event at the T M However, the thermogram for the DS1 and DS2 HCA II variants at pH 7.8 r eveal a predominant peak at 71 and 68 C respectively, but does not fit as precisely to a two state model due to minor transition peaks at lower temperatures ( Figure 5 2A ). A molten globule intermediate (<10% of the total population) that unfolds near the major unfolding transition for this DS1 HCA II variant has been reported earlier ( 172 ) Interestingly, the minor transition at 53 C at pH 7.8 for DS1 HCA II is not seen in the thermograms at pH 5.6, which reveals only a single t ransition event at ~60 C ( Figure 5 2C ). This suggests a non specific aggregation event involving deprotonated residues occuring at pH 7.8 and could be the molten globule dimer intermed iate that has been suggested to provide a nucleation site for further aggregation ( 214 ) Indeed, FPLC elution profiles and SDS

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77 PAGE analysis of affinity column purified the DS1 and DS2 HCA II variants samples at pH 7.8 do suggest very minor levels (<10%) of dimer formation and higher forms of oligomerization (data not shown). Thermodynamic analysis of the DS1 HCA II melting profiles ( 206 ) revealed properties that correlate well to previous tryptophan fluorescence experiments in similar conditions between DS1 and HCA II C206S ( 172 ) The additional 4.3 kcal mol 1 gain in free energy observed between the DS1 variant and wild type HCA II calorimetric data could be explained by the possible formation of a hydrogen bond between Ser206 and the backbone carbo nyl group of Val135 observed in the X ray crystallographic structure. The addition of Ser206, however, has no significant effect on the stability of the variant compared that to the native state ( T M < 1C; data not shown). This gain in free energy is consistent in the more acidic condition at pH 5.6, with a G T=52C of 9.1 kcal mol 1 The kinetic data revealed that DS1 and HCA II have comparable maximum k cat /K M values (Table 5 2 ). Similarily, th e rate of maximum proton transfer, k B for both enzymes was not significantly different and correlate well to the hydration activity of DS1 previously reported ( 172 ) The comparable catalytic rates are further supported by the absence of significant structural and solvent perturbations within the active site of DS1 compared to HCA II ( Figure 5 1C ) and additionally suggests that this variant can be implemented wi th mutations within the active site that have been shown to enhance the proton transfer rate of HCA II ( 32 ) The catalytic efficiency of DS2 HCA II was decreased two fold compared to wild type HCA II but with a similar proton transfer rate ( Figure 5 2B; Table 5 2). The structural consequences of the disulfide linkage between

PAGE 78

78 residues 6 and 11 in DS2 HCA II on the configuration of the water newtork in the active site c annot be established without an atomic model of this variant. 5. 5 Conclusions This study provides the first X ray crystallographic structure of an engineered disulfide bridge in HCA II ( Figure 5 1 ). Moreover, the 14 15 C enhanced denaturing temperature of this DS1 variant (Table 5 2; Figure 5 2) provides an active and stable CA that could better withstand the harsh conditions employed by current industrial protocols of atmospheric carbon sequestration (temperatures greater than 70 C and pH less than 6.0 ) ( 97 ) without loss of catalytic efficiency (Table 5 2 ; Figure 5 3E ). This designed disulfide linkage can be used in conjuction with other variants of HCA II from Chapter 4 that show e nhanced thermal stability via hydrophobic to hydrophilic surface mutations resulting in a gain of enthalpic contributions from hydrogen bonding ( 93 ) Interestingly, the addition of an additional disulfide bridge between residues 6 and 11 in HCA II did not have a stabilizing effect as compared to having only one linkage between residues 23 and 203. The enhanced rigidity around the N terminus created via the disulfide b ridge of the DS2 variant may account for its decreased catalytic activity and stability compared to DS1 HCA II This report demonstrates that rational design of a moderately conserved disulfide bridge into an isoform lacking this linkage can significantly enhance the stability of the enzyme without loss in the overall catalytic activity Other possible sites for engineering of disulfide linkages based on residue proximity and geometric requirements as calculated in Disulfide by Design ( 215 ) include: A la 153 S er 219, Phe 147 Pro 215, S er 56 Phe 179, Gln28 Gln 249 and Asp 190 Phe 260

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79 Table 5 1. X ray crystallographic data set and refinement statistics for DS1 HCA II PDB Accession Number 4HBA Wavelength () 1.5418 Spacegroup P2 1 Unit cell parameters (;) a = 42.3, b = 41.2, c = 71.6; 104.2 Total number of measured reflect ions 96044 Total number of unique reflections 23307 Resolution () 20.0 1.77 (1.83 1.77) R sym a (%) 7.0 (50.0) I / (I) 13.5 (2.7) Completeness (%) 98.2 (98.9) Redundancy 4.1 (4.2) R cryst b (%) 15.0 R free c (%) 18.9 Number of residues 257 Numbe r of protein atoms (including alternate conformations) 2301 Number of water molecules 203 r.m.s.d.: Bond lengths (), angles () 0.010, 1.304 Ramachandran statistics (%): Most favored, additionally allowed, generously allowed and disallowed regions 88. 4, 11.1, 0.5, 0.0 Average B factors ( 2 ): All, main chain, side chain, solvent 19.5, 18.7, 20.7, 29.2 r.m.s.d.: C () d 0.2 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensities. b R cryst = o F c o |) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refinement (5% of reflections for all data sets). Values in parenthesis are for the highest resolution shell.

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80 Table 5 2. Thermal stability an d catalytic m easurements for DS1 and DS2 HCA II Enzyme T M ( C) a k cat ex /K eff S (M 1 s 1 ) b k B 1 ) b pH 7.8 pH 5.6 wild type HCA II 5 7.1 ( 0.1) 45.4 ( 0.1) 120 ( 20) 0.8 ( 0.1) DS1 (A23C/L203C/C206S) 71.0 ( 0.1) 59.6 ( 0.1) 1 31 ( 20) 0.5 ( 0 .1) DS2 (G6C/N11C/A23C/L203C/C206S) 68.6 ( 0.1 ) N/D 50 ( 20) 0.8 ( 0.2) a Denaturation temperature as determined by DSC. b The rate c onstants are determined by fitting Eqs. (4 6) and ( 4 7) to the data of Figure 5 3

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81 Figure 5 1. X ray crystallograp hic model of DS1 HCA II. A) Cartoon view of DS1 (green) with the Zn 2+ metal shown as a magenta sphere. Coordinating histidines, His64 and the disulfide linkage between residues 23 and 203 are shown in stick model. B) Zoomed in view of disulfide linkage and comparison of C backbone to wild type HCA II (PDB: 3KS3) (gray ribbon). C) Active site of DS1 with corresponding residues and water molecules labeled.

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82 Figure 5 2. Thermograms for DS1 DS2 and wild type HCA II Denaturing curves for DS1 HCA II at A) p H 7.8 and B) 5.6; wild type HCA II at C) pH 7.8 and D) pH 5.6; E) DS2 HCA II at pH 7.8 Experimental data is shown in black with the fit to the data shown in red.

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83 Figure 5 3 Catalytic Activity of the DS1 and DS2 HCA II variants A) The catalytic effi ciencies and B) proton transfer rates for DS1 HCA II (closed square) and wild type HCA II (open circle) The same are shown for DS2 HCA II (closed circle) in C) and in D) assuming a single ionization constant (closed circles) and double ionization constant s (open squares).

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84 CHAPTER 6 ADDITION OF AROMATIC CLUSTERS 6.1 Overview The presence of aromatic clusters has been found to be an integral feature of many proteins isolated from thermophilic microorganisms. Residues found in aromatic cluster interact via the weakest interactions involved in protein stability. The lone aromatic cluster in HCA II is centered on Phe 226 with the surrounding aromatics Phe 66, Phe 95 and Trp 97 located 12 posterior th e active site; a location which could facilitate proper protein folding and active site construction. The role of Phe 226 in the structure, catalytic activity and thermostability of HCA II was investigated via site directed mutagenesis of three variants (F2 26I/L/W) into this position. Additionally, superimposition of the crystallographic structure of HCA IV onto HCA II revealed a cluster of aromatic resiudes unique to HCA IV (See Figure 3 3). These mutations (termed Aros HCA II : L47F/V49F/I146/L212 F ) were in corporated in addition to the lone aromatic cluster centered on Phe226 in HCA II. This chapter emphasizes the importance of t he delicate arrangement of the weak C interactions among aromatic clusters in overall protein stability. 6.2 Methodology 6.2. 1 Protein Expression and Purification Primers from Invitrogen and the Stratagene QuikChange II kit were used for site directed mutagenesis in the preparation of HCA II cDNA with the individual F226I/F226L/F226W mutations as well as the L47F/V49F/I146/L212F HCA II variant (Aros) from an expression vector consisting of the enzyme coding region ( 141 )

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85 Verification of cDNA sequence, protein expression and purification a re identical to that detail in Section 4.2.1. The Aros HCA II variant expresses about half as well as HCA II, and is susceptible to oligomerization upon protein purification as evidenced via visual inspection of SDS PAGE gel (data not shown). 6.2.2 X ray Crystallography C rystal tray s were prepared for the Phe 226 HCA II variants with a 1:1 ratio of the reservoir solution and the protein sample (10 mg/mL) in 5 L drops The reservoir solutions contained 500 L of solution between 1.4 1.6 M sodium citrate, 50 mM Tris, pH 8.0 9.0. The crystals formed in approximately one week via the hanging drop diffusion method. The Aros HCA II variant has resisted crystal f ormation in several Hampton crystallization screens using varying protein concentrations. This is likely due to its preference to aggregate, as evidenced via the multimeric states as stated above. Diffraction data sets for the Phe 226 variants were collect ed on an in house Rigaku R Axis IV ++ image plate detector with a RU H3R rotating Cu anode (K = 1.5418 ) operating at 50 kV and 22 mA. The X rays were focused using Osmic optics, followed by a helium purged beam path. The crystal to detector distance w as 80 mm Each image was collected for five minutes with 1 oscillations HKL2000 ( 193 ) was used to integrate, merge and scale the data sets. The variants crystallized in the monocli nic space group P2 1 and diffracted to resolutions of 2.05, 1.63 and 1.70 for F226I, F226L and F226W, respectively. The diffraction and refinement statistics for all of the variants are summarized in Table 6 1. Initial phases of the variants were calculated using molecular replacement using the wild type HCA II structure (PDB: 3KS3; ( 68 ) ) with 5% of the reflections set aside for R free calculations. PHENIX.REFINE ( 194 ) was used in cycles of restrained refinement of

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86 the molecular model, alternating with manual building using COOT ( 195 ) Initial F o F c difference maps revealed negative density around the phenyl ring (present in the molecular replacement structure) at po sition 226 for the Leu and Ile variants, whereas F226W showed positive density that extended further away from the phenyl ring. Subsequent refinements showed excellent electron density in the 2F o F c maps for the respective variants at this position ( Figu re 6 1 ) The final R crys t and R free values are shown in Table 6 1. MOLPROBITY ( 216 ) was used to assess the quality of the final model. All structural figures were cr eated in PyMOL ( 205 ) Experiment al data and structural coordination have been deposited with the Protein Data Bank under the accession numbers 4L5U, 4L5V and 4L5W for F226I, F226L and F226W, respectively. 6.2.2 Differential Scanning Calorimetry Thermograms for the Phe 226 and Aros HCA II variants were collected and anlayzed in triplicate at pH 7.8 as outlined in Section 4.2.2. 6.2.3 Kinetic Studies The catalytic rates of the Phe 226 and Aros HCA II variants were analyzed using 18 O labeled mass spectrometry as outlined in Section 4.2.3. 6.3 Results 6.3.1 X ray Crystallography The Phe 226 HCA II variants crystallized in the monoclinic P2 1 space group with approximate unit cell dimensions of a = 42, b = 41 and c = 72 , = 104 and diffracted to medium resolution (1.63, 1.70 and 2.05 for F226 L, F226W and F226I, respectively). The final R cryst /R free values were 13.0/15.3, 14.2/18.2 and 15.4/20.1 % for F226L, F226W and F226I, respectively. A summary of the collected diffraction and final

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87 refinement statistics is shown in Table 6 1 The intial F o F c maps for each variant confirm ed the successful amino acid substitutions at residue 226 in HCA II ( 217 ) A linear least square fit of the C a toms in the Phe 226 variants to HCA II (PDB: 3KS3; ( 68 ) ) revealed little structural variation (r.m. s.d. ~0.10 ; Table 6 1 ). There were, however, minor local rearrangements (~0.2 shift in C positioning) of Phe 95 and Trp 97 within the secondary CO 2 binding pocket of the F226W variant. These structural conformational changes could be contributed to erro r within the positioning of the atomic coordinates (~10% of the high resolutions) and are not considered to be of significance. It is important to note that the loss of C H bonds in the Phe 226 variants does not alter the configuration of the side and m ain chain atoms in the aromatic cluster ( Figure 6 1) Interestingly, the active site residues and the proton shuttle water network of the Phe 226 variants were essentially identic al to that of HCA II (< 0.1 r.m.s.d.), indirectly indicating that these varian ts and the native enzyme should have comparable catalytic rates to one another. The proton shuttle residue H is 64 ( 23 ) was seen to be in a dual wards the active site) in the F226I variant. These variable conformations among crystallographic structures of HCA II are not an uncommon occurrence and are dependent on the crystallization conditions ( 20 29 32 136 206 ) 6.3.2 Thermal Stability The thermal unfolding transitions of the Phe 226 and Aros HCA II variants at pH 7.8 were studied in triplicate utilizing DSC The peaks were fitted assuming a simple two state transition, with further complexity (e.g., multi ple transition peaks) added if

PAGE 88

88 necessary ( Figure 6 2 ). These peaks were calculated to be endothermic and were centered at the denaturing temperature (T M ), with the results summarized in Table 6 2 Interestingly, the T M for the F226I/L variants was 3 4 C lower than that of the native enzyme (~53 versus 57 C respectively; Figure 6 2A/B; Table 6 2 ; ). The thermogram for the F226W variant displayed a broad dissociation peak consistent with three intermediate states: one that has a T M s imilar to that of F22 6I/L at 54 C, one similar to that of HCA II at 56 C and one at a lower temperature than observe d for the other variants at 51 C ( Figure 6 2C; Table 6 2 ). However, the H M / H vH ratios for the F226W variant at the higher denaturation temperature are indicative of oligomeric aggregation. Calculation of the H M / H vH ratios for F226I and F226L are indicative of a single unfolding transition and are comparable to that o f wild type HCA II ( 217 ) Comparison of the calculated G ( Eq. 4 1) at 55.1 C for the variants and HCA II shows the energy difference between these variants are ~2 3 kcal mol 1 ( 217 ) an energy dissociation that can be correlated to C ( 218 ) The ab sence of local reconfiguration and decreased thermostability of the Phe 226 variants compared to that of HCA II suggests that a phenyl ring at position 226 makes important enthalpic contributions to the overall stability of the enzyme. The thermograms obtained for Aros HCA II were indicative of a single unfolding transition, similar to HCA II, with a T M of ~84 C ( Figure 6 2D; Table 6 2). This is an exceptional increase in thermal stability compared to wild type HCA II ( T M > 25 C) If the Aros HCA II variant were to be expressed in nature, it would be classified as a hyperthermophilic enzyme (optimal growth temperature >80 C).

PAGE 89

89 6.3.3 Catalytic Activity The pH profiles for the two rate constants, k cat /K M and R H2O were det ermined by measuring the exchange of 18 O label between CO 2 and H 2 O via mass spectrometry. The rates of catalyzed interconversion between CO 2 and HCO 3 as measured by k cat /K M (Eq. 4 6 ; Figure 6 3 A ; Table 6 2 ) for the Phe 226 variant s were comparable to that of HCA II. F226I showed the most similarit y in catalytic efficiency to HCA II (~120 M 1 s 1 ) whereas F226L was slightly higher at 140 M 1 s 1 and F226W slightly lower (87 M 1 s 1 ). The catalytic efficiency of the Aros HCA II variant ( Figure 6 3C ; Table 6 2) was found to be reduced by three fold compared to wild type HCA II (~40 M 1 s 1 ). An X ray crystallographic structure is needed to assess th is loss of catalytic activity These two to three fold loss in catalytic activity however, are not considered to be significant. The measured rate constant for proton transfer through the water network in the active site, k B from Eq. 4 7 reveals a similar pattern as that seen in the k cat /K M measurements ( Figure 6 3B ; Table 6 2 ). Both the F226I and F226L variants show comparable k B values to that of wild type 1 ) whereas F226W had a slight 1 As discussed with the catalytic efficiency measurements, these variations in rat es for the Phe 226 variants are not considered to be of significance. The measured k B rate for Aros HCA II, however, revealed a proton transfer rate that was six fold decreased (0.2 s 1 ) compared to HCA II ( Figure 6 3 D; Table 4 2). This loss in the proton transfer rate and the catalytic efficiency of Aros HCA II suggest disruption in the activ e site configuration of the residues and/or water molecules involved in catalysis.

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90 6.4 Discussion These studies investigated the significance of the proximity of the lone aromatic cluster in HCA II to the active site (~12 from the zinc metal) in the cata lytic efficiency and thermal stability, as well as the biophysical effects of adding an additional aromatic cluster located directly posterior the active site X ray crystallographic analysis revealed that site directed mutagenesis of position 226 to a con served human variant (F226L) and other non conserved residues (F226I/W) did not significantly alter the aromatic cluster ( Figure 6 1 ) o r the active site of the variants Kinetic measurements via 18 O mass spectrometry confirmed the overall catalytic rate an d proton transfer of the Phe 226 variants did not significantly change compa red to that of wild type HCA II ( Table 6 2 ). DSC studies revealed, however, some reduction in the denaturing temperature ( T M 3 C ) o f the variants as compared to wild type HCA II ( Table 6 2 ). The source for this destabilization in the Phe 226 variants may be explained via disruption of weak C Phe66 Phe 95 a nd Trp 97 ( Figure 6 1 ). These weak hydrogen bonds between soft acids and bases are highly orientation and spatially dependent (ideally perpendicular and 3.2 3.8 distance) and contribute ~2 kcal mol 1 enthalpically ( 151 152 175 179 188 ) Substitution of Phe 226 with either I le/Leu results in complete loss of the meta and para interactions with Phe 66 and Phe 95 The methyl group of F226I, however, is seen to be within 3.3 of the center of the indole gro up o f Trp 97 at an angle of 112 suggesting a potential weak C distance relationships (4.5 and 4.7 ) with Trp 97 can be found between the terminal carbons, the closer of the two being nearly perpendicular The interaction between Phe 95 i s restored

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91 in the F226W mutant at a closer distance (3.5 ), but a larger angle from ideal (115), due to molecular crowding of the introduced indole ring. The interaction of F226W with Phe 66 and Trp97 is also weakened with increased distances (>4.4 ) and angles (~ 120) for C The structural identity of t he Phe 226 variants compared to wild type HCA II cou pled with their decreased thermal stability suggests that the loss of C with Phe 66 and Phe 95 (for F226I/L) decreases the denaturatio n temperature by ~4 C The broader transition phase seen with F226W that restores stability up to near native levels emphasizes the apparent importance of maintaining these weak interactions among these aromatic cluster residues. This notion is further ex tenuated when considering the significant increase in thermal stability in the Aros HCA II variant, containing an additional aromatic cluster. Caution must be used, however, in that the addition of this hydrophobic core in HCA II raises issues with protein expression, solubility and catalytic activity. 6.5 Conclusions These studies suggests that while the aromatic cluster centered on Phe 226 in HCA II does not co ntribute to enzymatic activity it does play an important role in protein stability (Table 6 2) The importance of weak C interactions in the global stability of HCA II was illustrated as the absence or distortion of these bonds in the Phe 226 va ri ants all resulted in a decrease in thermal stability of ~4 C as compared to wild type HCA II Extenuating this idea, the addition of an aromatic cluster that is uniquely found in HCA IV to HCA II should provide a source for thermal stability. Th e resultant was a hyperthermophilic Aros HCA II variant that saw an increase in melting temperature by

PAGE 92

92 over 25 C compared to the na tive enzyme. The addition of this hydrophobic core, however, had detrimental effects on the expression, solubility and catalytic activity of Aros HCA II and, therefore, may not be an ideal candidate for biomedical and/or industrial usage. Site directed mut agenesis of the Phe 226 aromatic cluster in Aros HCA II to replicate the hydrophobic core found in HCA IV (consisting mostly of Leu and Ile residues) would provide interesting details in understanding if the unique aromatic cluster in HCA IV promotes its ex ceptional resistance to denaturation in 5% SDS ( 171 ) The catalytic activity of Aros HCA II could be restored with the inclusion of N67Q and/or Y7F active site mutations, but with an expected reduction of thermal stability, as discussed in Section 4.4.

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93 Table 6 1. X ray crystallographic data set and refinement statistics for Phe 226 HCA II v ariants F226I F226L F226W PDB Accession Number 4L5U 4L5V 4L5W Wavelength () 1.5418 1.5418 1.5418 Spacegroup P2 1 P2 1 P2 1 Unit cell parameters (;) a = 42.3, b = 41.4, c = 71.5; 104.3 a = 42.4, b = 41.4, c = 72.2; 104.4 a = 42.1, b = 41.3, c = 72.0; 104.3 Total number of measured reflections 103017 107591 77729 Total number of unique reflections 15372 28174 24828 Resolution () 20.0 2.05 20.0 1.63 20.0 1.70 R sym a (%) 11.1 (42.7) 6.8 (35.2) 4.1 (44.2) I / (I) 13.5 (3.9) 37.2 (5.1) 23.3 (2.6) Completeness (%) 99.0 (98.8) 92.2 (85.0) 92.8 (98.8) Redundancy 6.7 (6.5) 3.8 (3.8) 3.1 (3.0) R cryst b (%) 15.4 13.0 14.2 R free c (%) 20.1 15.3 18.2 Number of residu es 257 257 257 Number of protein atoms (including alternate conformations) 2300 2298 2293 Number of water molecules 209 338 290 r.m.s.d.: Bond lengths (), angles () 1.297, 0.011 1.333, 0.010 1.313, 0.010 Ramachandran statistics (%): Most favored, al lowed, outliers 97.2, 2.8, 0.0 96.9, 3.1, 0.0 96.9, 3.1, 0.0 Average B factors ( 2 ): All, main chain, side chain, solvent 9.9, 5.9, 10.3, 27.4 21.2, 16.2, 21.7, 34.2 29.0, 24.3, 30.3, 39.8 r.m.s.d.: C () d 0.10 0.08 0.14 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensities. b R cryst o F c o |) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refinement (5% of reflections for all data sets). Values in parenthesis are for the highest resolution shell.

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94 Table 6 2. Thermal st ability and catalytic m easurements of the Phe 226 and Aros HCA II v ariants Enzyme T M ( C) a k cat ex /K eff S (M 1 s 1 ) b k B 1 ) b wild type HCA II 5 7.1 ( 0.1) 120 ( 20) 1.4 ( 0.2) F226I 53.0 ( 0.1) 1 18 ( 5) 1.4 ( 0.2) F226L 53.2 ( 0.1 ) 139 ( 4) 1.3 ( 0.2) F226W 51.0 ( 0.2 ) 54.1 ( 0.7 ) 56.1 ( 0.2 ) 87 ( 3 ) 1.0 ( 0.3 ) Aros HCA II (L47F/V49F/I146F/L212F) 83.7 ( 0.1 ) 38 ( 1 ) 0.21 ( 0.04 ) a Denaturation temperature as determined by DSC. b The rate c onstants are determined by fitting Eqs. (4 6) and ( 4 7) to Figures 6 3 and 6 4

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95 Figure 6 1. Overlay of the X ray crystallographic structures of the Phe 226 variants. The aromatic cluster residues are shown in stick model and as labeled. F226I: orange; F226L: pink; F226W: yellow. Potential C H bonds are indicated with a black dashed line.

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96 Figure 6 2. Thermograms for the Phe 226 and Aros HCA II variants. A) F226I B) F226L C) F226W and D) Aros HCA II Experimental data is shown as black line whereas fit to to the data are shown in red.

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97 F igure 6 3. Catalytic activity of the F226 and Aros HCA II variants. A) Data for each variant are represented throughout as follows: HCA II, solid square; F226I, open triangle; F226L, cross; F226W, open circle. Calculation of k cat /K M (M 1 s 1 ) versus pH fr om Eq. 4 6. B) The pH profile for the rate constant R H2O /[E] 1 ) used in calculation of the proton transfer constant, k B via Eq. 4 7 for the Phe 226 variants C) Catalytic efficiency of Aros HCA II (solid circle) and D) pH profile showing the rates for R1/[E] in closed circles and R H2O /[E] in open squares

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98 CHAPTER 7 INTRODUCTION OF PROLINE RESIDUES 7.1 Overview The introduction of proline residues has been proposed as a protein stabilization method such that the entropy of unfolding is decreased ( 180 ) Proline, which can adopt only a few configurations and restricts the conformations of the preceding residues ( 181 ) has the lo west conformational entropy of all the amino acids. Thus, mutations of Xaa protein, assuming the engineered residue does not introduce unfavorable strains in the protein structure. Residues Lys170, Glu 234 and Leu240 in HCA II ( Figure 3 4) are the pr turns with no obvious and thus can naturally adopt the required dihedral angles seen in the i +1 position in a turn ( 185 ) The mutation L240P has already been s hown in Chapter 4 to impose thermal stability upon HCA II without any detrimental effects on the catalytic activity of the enzyme. This chapter will highlight the biophysical consequences of introducing a proline residue at sites 170 and 234 in HCA II. 7.2 Methodology 7.2.1 Protein Expression and Purification Primers from Invitrogen and the Stratagene QuikChange II kit were used for site directed mutagenesis in the preparation of HCA II cDNA with the individual K170P and E234P mutations from an expression v ector consisting of the enzyme coding region ( 141 ) Verification of cDNA sequence, protein expression and purification are identical to that detail in Section 4.2. 1.

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99 7 .2.2 X ray Crystallography C rystal tray s were prepared for the E234P HCA II variant with a 1:1 ratio of the reservoir solution and the protein sample (10 mg/mL) in 5 L drops The reservoir solutions contained 500 L of 1.4 M sodium citrate, 50 mM Tri s, pH 7.8. The crystals formed in approximately one week via the hanging drop diffusion method The K170P HCA II variant has resisted all crystallization attempts using the Gryphon Screening Robot (Art Robbins) and crystallization screens from Hampton Rese arch. Diffraction data sets for E234P were collected on an in house Rigaku R Axis IV ++ image plate detector with a RU H3R rotating Cu anode (K = 1.5418 ) operating at 50 kV and 22 mA. The X rays were focused using Osmic optics, followed by a helium purged beam path. The crystal to detector distance w as 80 mm Each image was collected for five minutes with 1 oscillations HKL2000 ( 193 ) was used to integrate, merge and s cale the data sets. The variant crystallized in the monoclin ic space group P2 1 and diffracted to a resolution of 1.90 . The diffraction and refinement statistics for E234P HCA II are summarized in Table 7 1. Initial phases of the variants were calculated using molecular replacement using the wild type HCA II struc ture (PDB: 3KS3; ( 68 ) ) with 5% of the reflections set aside for R free calculations. PHENIX.REFINE ( 194 ) was used in cycles of restrained refinement of the molecular model, alternating with manual building using COOT ( 195 ) Initial F o F c difference maps revealed negative density at position 234 Subsequent refinements showed excellent electron density in the 2F o F c maps for the E234P mutation ( Figure 7 1B ) The final R crys t and R free values are shown in Table 7 1. MOLPROBITY ( 216 ) was used to assess the quality of the final model. All structural figures were created in PyMO L ( 205 )

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100 7.2.3 Differential Sca nning Calorimetry DSC studies were conducted and analyzed in triplicate at pH 7. 8 as described in Section 4.2.3 to study the thermal stability of E234P and K170P HCA II. 7.2.4 Catalytic Activity 18 O mass spectrometry studies were carried out at 20 C as ou tlined in Section 4. 2 4 to measure to catalytic activity of E234P and K170P HCA II. 7.3 Results 7.3.1 X ray Crystallography The E234P HCA II variant ( Figure 7 1A) crystallized in the monoclinic P2 1 space group with unit cell dimensions of a = 42 .2 b = 40. 8 and c = 71.4 , = 104 .2 and diffracted to a resolution of 1.90 The working R work /R free values are 17.3 and 23.0 % respectively. A summary of the collected diffraction and final refinement statistics is shown in Table 7 1. The initial F o F c maps confirmed the succ essful amino a cid substitutions at residue 234 in HCA II with the final 2F o F C map showing adequate Figure 7 1B). Superimposition of wild type HCA II onto that of E234P displayed comparable configurations for the loop in which the mutation is located (r.m.s.d. ~0.2 ) as well as for active site residues and water molecules ( Figure 7 1C), suggesting the catalytic activity of E234P should not be compromised. Interestingly, the resulting F o F c map after refinement with addition of Zn 2+ in the active site resulted in a linear bilobal distribution of positive electron density near the metal ion that is best explained by the presence of CO 2 ( Figure 7 1C) in 0.86 occupancy. The substrate molecule contains comparable B factors (23.0 2 ) as to the proton transfer water network molecules (26.6 2 ) in t he active site. However, the B factors for W1 and W2 in the E234P HCA II active site are relatively large compared to

PAGE 101

101 those for W3A and W3B (~40 versus ~15 2 respectively ) This may refle ct the thermal fluctuations that are more commonly experienced with the water molecules in the early stages of proton transfer as opposed to those deep in the hydrophilic pocket. 7.3.2 Thermal Stability The thermograms for the E234P and K170P HCA II varia nts were obtained in triplicate before being averaged, buffer subtracted and baseline corrected. The major transition peak for both variants displayed a single unfolding dynamic ( Figure 7 2). The E234P HCA II variant displayed a T M of ~58 C ( Figure 7 2A), a modest increase in thermal stability compared to wild type HCA II (~57 C, Table 7 2) whereas the K170P variant resulted in a loss of stability to about ~56 C ( Figure 7 2B). The insignificant and decrease in melting temperatures of the E234P and K170P HCA II variants as compared to the native enzyme reveal that introduction of proline residues at these positions are not a source of thermal stability as seen with the L240P HCA II variant (Chapter 4). 7.3.3 Kinetics The pH profiles for the two rate consta nts, k cat /K M and R H2O were determined by measuring the exchange of 18 O label between CO 2 and H 2 O via mass spectrometry. The rates of catalyzed interconversion between CO 2 and HCO 3 as measured by k cat /K M (Eq. 4 6; Table 7 2 ) for both E234P ( Figure 7 3A) a nd K170P ( Figure 7 3C) HCA II were decreased by two fold compared to wild type HCA II (60 versus 120 M 1 s 1 respectively). These two fold fluctuations, however, are not considered to be significant. Inspection of the solved E234P HCA II crystallographic structure reveals no evidence for the decreased catalytic efficiency.

PAGE 102

102 The measured rate constant for proton transfer through the water network in the active site, k B from Eq. 4 7 reveals an ~three fold increase for the E234P HCA II variant (3.1 s 1 ) com pared to wild type HCA II ( Figure 7 3B) whereas for the K170P variant the k B is comparable to the native enzyme (1.4 s 1 ; Figure 7 3D; Table 7 2). These increased proton transfer rates in the K170P and E234P HCA II variants may be associated with the de creased pK a values for His64 dropping an entire pH unit compared to the wild type enzyme (Table 7 2) A deprotonated His64 would more readily accept a proton from W2, promoting faster proton transfer rates. 7.4 Discussion Introduction of proline residues at key locations throughout a protein chain has been shown to enhance the thermal stability of several enzymes including the L240P HCA II variant. The E234P and K170P HCA II variants are unlike the L240P mutation, however, as these variants replace charged ionic residues with prolines, as compared to the hydrophobic to hydrophilic substitution described in Section 4.3. The absence of any intra or intermolecular interactions at positions 170 and 234 in the high resolution crystallographic structure (PDB: 3 KS3) suggests that these sites should rigidify their respective loop regions, resulting in an increase in thermal stability. The thermograms obtained from DSC, however, showed that the E234P mutation ( Figure 7 2A) had an insignificant increase in the melti M < 1 C; Table 7 2). The melting temperature for the K170P variant ( Figure 7 2B) actually decreased about 1 C compared to the wild type enzyme (Table 7 2). The source of this destabilization is unknown without an available cry stal structure, but it must be noted that residue 170 is directly across the from the highly acidic 230 240 loop in HCA II (See Chapter 8) which may provide a potential site for charged interactions. The lackluster increase in thermal

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103 stability of the E234 P HCA II variant compared to that seen with L240P HCA II may be explained in with the relative decreased surface hydrophilicity. As discussed in Section 4.4, the L240P mutation decreases the hydrophobicity of the HCA II surface, thereby allowing water mole cules to interact with previously inaccessible sites. The E234P mutation does not decrease the hydrophobic nature of the enzyme surface but does provide a source of loop rigidity. The catalytic measurements of the K170P and E234P HCA II variants revealed an overall two fold decrease in the catalytic efficiencies compared to wild type HCA II (k cat /K M of 60 M 1 s 1 ; Table 7 2). While these decreases in kinetic activities are considered non significant, the source for this catalytic loss from the crystallogr aphic structure is not evident. Interestingly, while the catalytic efficiencies of both variants are compromised by two fold, the rate of proton transfer (k B ) were either comparable to wild type (for K170P) or three fold faster (E234P; see Table 7 2). This increase rate of proton transfer could be due to the lowered pK a values measured for His64 (Eq. 4 7) but further spectrophotometric measurements ( 37 ) are needed to confirm these results. 7.5 Conclusions The introduction of proline residues at positions 1 70 and 234 in HCA II was shown to have a detrimental and modest effect in the thermal stability of the enzyme, unlike that observed with the L240P mutation (Chapter 4). Additionally, mutation at these sites had a negative effect on the catalytic efficienci es of the variants, decreasing it by two fold as compared to wild type HCA II. As such, the K170P and E234P HCA II variants are not considered prime candidates for industrial and biomedical use. It is interesting to note, however, that the catalytic activi ty of HCA II is not solely dependent on the composition and configuration on the residues within the active site, but that the

PAGE 104

104 residues on the peripheral of the active site help define the kinetic parameters of the enzyme. Additionally, the E234P HCA II s tructure is the first crystallographic structure that shows CO 2 bound in the native active site without having to pump in ~15 atm of the gas into the crystals. Molecular dynamic simulations utilizing the previously solved CO 2 /HCO 3 bound structures ( 54 59 ) and the E234P HCA II structure may provide unique insight into the molecular mechanism of CA catalysis ( Figure 7 1D ). An oxygen atom of the CO 2 binds within 3.5 of the backbone amide group of Thr200 ( Figure 7 1D; yellow), which then moves closer to be within 3.2 ( Figure 7 1D; cyan). The distal oxygen atom of CO 2 is then moved closer towards the zinc metal while the proximal oxygen moves to within 2.9 of the Thr200 backbone nitrogen ( Figure 7 1D; green) before nucleophilic attack by the ZnOH (2.8 ) catalyzing the hydration into bicarbonate (cyan). Further crystallographic data at higher resolution of CO 2 bound i n E234P HCA II could provide a deeper knowledge into the catalytic mechanism of HCA II.

PAGE 105

105 Table 7 1. X ray crystallographic data set and refinement statistics for E234P HCA II Wavelength () 1.5418 Spacegroup P2 1 Unit cell parameters (;) a = 42.2 b = 40.8 c = 71.4 ; 104.2 Total number of measured reflections 66891 Total number of unique reflections 17538 Resolution () 20.0 1.9 (1.96 1.90)* R sym a (%) 9.6 (49.7) I / (I) 9.4 (2.2 ) Completeness (%) 93.8 (91.2 ) Redundancy 3.8 (4.0 ) R work b (%) 17.3 R free c (%) 23.0 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensiti es. b R work o F c o |) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refinement (5% of reflections for all data sets). Values in parenthesis are for the highest resolution shell.

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106 Table 7 2 Therma l stability and catalytic m easurements for E234P and K170P HCA II Enzyme T M ( C) a k cat ex /K eff S (M 1 s 1 ) b k B 1 ) b pK a ZnOH b pK a His64 b wild type HCA II 5 7.1 ( 0.1) 120 ( 20) 1.2 ( 0.2) 6.9 ( 0.1) 7.2 ( 0.1) E234P 57.9 ( 0.1) 56 ( 2) 3.1 ( 0.6) 7.2 ( 0.1) 6.2 ( 0.1) K170P 55.7 ( 0.1 ) 63 ( 3) 1.4 ( 0.3) 6.7 ( 0.1) 6.4 ( 0.1) a Denaturation temperature as determined by DSC. b The rate c onstants are determined by fitting Eqs. (4 6) and ( 4 7) to Figures 7 3

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107 Figure 7 1. Crystallographic structure of E234P HCA II. A) Cartoon view of E234P HCA II (green) with the Zn 2+ metal ion shown as magenta sphere; the coordinating His residues, Pro234 and CO 2 are shown as sticks. B) 2F o F c map contoured at 0.9 (gray mesh) showing the Pro234 mutation. C) Active site structure with the CO 2 (green stick) electron density shown in gray mesh contoured at 1.2 Residues and water molecules are as labeled. D) Superimposition of previously determined CO 2 bound structu res (PDB: 3D92: yellow; 3U7B: cyan) revealing detailed coordinates of substrate catalysis.

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108 Figure 7 2. Thermograms of A) E234P and B) K170P HCA II. The experimental data is shown as a black line with the fit shown in red.

PAGE 109

109 Figure 7 3. Catalytic acti vities of E234P and K170P HCA II. A) Catalytic efficiency of E234P HCA II (closed circles). B) pH profile of E234P HCA II with R1/[E] (Eq. 4 6) shown as closed circles and R H2O /[E] as open squares. C) and D) are identical to A) and B) except for K170P HCA II.

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110 CHAPTER 8 TRUNCATION OF SURFACE LOOPS 8.1 Overview A current working hypothesis for the source of a thermophilic proteins stability is the extreme surface rigidity created via surface loop truncations and/or deletions This is supported by numerous e xperimental data that includes frequency domain fluorometry and anisotropy decay ( 186 ) hydrogen deuterium exchange ( 187 189 ) and tryptophan phosphorescence ( 190 ) In Chapter 7, the rigidity of the surface loop in HCA II at position 234 via introduction of a proline residue was seen to increase along with a corresponding increase in thermal stability. This could be explained by the observation that loop s are usually the regions with the highest thermal factors in a protein crystal and, as such, are likely to unfold first during thermal denaturation. Loop truncation on several mesophilic proteins has increased the melting temperature in previous studies ( 167 191 ) and will be extended to include HCA II. The extended loop region at residues 230 240 is a good candidate for truncation ( Figure 3 5; orange segment ) as it : 1) has a higher average C B factor (thermal fluctuation) than the rest of enzyme ; 2) is not involved in any intramolecular hydrogen bond networks ; 3) has tw o highly hydrophobic residues (Phe 231 and V al 240) that are solvent exposed and 4) the same loop region was found to be delet ed in the recently characterized hyperthermophilic CA from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) ( 203 ) Furthermore, the carbonyl group of L eu 229 is sufficiently close to the amide group of M et 241 (2.8 ) that it can participate in a peptide bond without signifi cant backb one alteration ( Figure 3 5; green segment ). The biophysical effects on the thermal stability, structure and catalytic activity from removing this acidic

PAGE 111

111 loop located on the peripheral of the active site of HCA II will be investigated in this chapter. 8.2 M ethodology 8.2.1 Protein Expression and Purification The plasmid cDNA expressing the loop truncation between residues 230 and 240 240 HCA II) was created utilizing the PCR mediated plasmid DNA deletion method ( 219 ) In short, two primers were designed (Primers A and B) with Primer A being designed as the reverse complement of a sequence corresponding to 20 bases upstream of the plasmid DNA to be deleted followed by 20 bases eq ual to the downstream sequence. Primer B was designed in the same fashion but corresponding to the complementary strand; that is, the sequence of Primer B was identical to the plasmid primary lacking the sought deletion. The truncated plasmid cDNA was then transformed, expressed and purified as outlined in Section 4.2 1. 8.2.2 X ray Crystallography C rystal tray s were prepared for the 230 240 HCA II variant with a 1:1 ratio of the reservoir sol ution and the protein sample (55 mg/mL) in 5 L drops The reservoir solutions contained 500 L of solution between 1.6 sodium citrate, 100 mM Tris, pH 7.8. The crystals formed in approximately o ne week via the hanging drop diffusion method. Diffraction data sets for the 230 240 HCA II variant was collected on the F1 beamline at Cornell High Energy Sychrotron Source ( CHESS F1; = 0.9177 ) on an ADSC Q 270 detector using the microfocused beam. The crystal to detector distance w as 150 mm Each image was collected for 10 seconds with 0.5 oscillations HKL2000 ( 193 ) was used to integrate, merge and s cale the data sets. The variant crystallized in

PAGE 112

112 the orthorhombic space group P2 1 2 1 2 1 and diffracted to a high resolution of 1. 35 The diffractio n and refinement statistics for 230 240 HCA II are summarized in Table 8 1. Initial phases for 230 240 HCA II were calculated using molecular replacement from an in silico model of the truncated variant generated from the wild type HCA II structure (PDB: 3KS3; ( 68 ) ) with 5% of the reflections set aside for R free calculations. PHENIX.REFINE ( 194 ) was used in cycles of restrained refinement of the molecular model, alternating with manual building using COOT ( 195 ) Initial 2 F o F c difference maps revealed excellent density around the peptide backbone between residues 229 and 241. The working R work and R free values are 20.8 and 24.5%, respectively (Table 8 1). All structural figures were created in PyMOL ( 205 ) 8.2.3 Differential Scanning Calorimetry DSC studies were conducted and analyzed in triplicate at pH 7.8 as described in Section 4.2.3 to study the thermal stability of 230 240 HCA II. 8.2.4 Catalytic Activity 18 O mass spectrometry studies were carried out at 20 C as outlined in Section 4. 2 4 to measure to catalytic activity of 230 240 HCA II. 8.3 Results 8.3.1 X ray Crystallography The 230 240 HCA II variant crystall ized in the monoclinic P2 1 2 1 2 1 space group with unit cell dimensions of a = 4 3.1 b = 69.0 and c = 42.0 ( = 90 ) and diffracted to a resolution of 1.35 The working R work /R free values are 20.8 and 24.5 % respectively. A summary of the collected diffr action and final refinement statistics is shown in Table 8 1. The initial 2 F o F c maps following molecular replacement confirmed the successful

PAGE 113

113 deletion of residues 230 240 in HCA II Superimposition of wild type HCA II onto that of 230 240 HCA II ( Figur e 8 1A) revealed the engineered peptide bond between Leu229 and Met241 shifts the positioning of the sidechains of those residues as compared to the wild type structure containing the loop. This flexibility is most emphasized in Met241, which may be in a d ual configuration compared to the single, low B factor state in the wild type structure. Comparison of the 230 240 HCA II active site to the wild type structure ( Figure 8 1B) reveals very little deviation in the positioning of the active site residues. However, there are extreme variations in the proton transfer water network in the 230 240 HCA II variant ( Fi gure 8 1B; red spheres) versus that seen in the native enzyme (pink spheres). Both W1 and W2 have been shifted ~0.2 in the variant active site whereas W3B, which interacts with Asn62 and Asn67 in the native structure, has been shifted 2.8 in the 230 2 40 HCA II active site so that it only interacts with the amine group of Gln92 and W4 (a water molecule that is seen to interact with W1 and Gln92 in the native structure; see Figure 8 1B). The W4 molecule itself has been shifted 2.2 in the 230 240 HCA I I active site as to interact only with W1 due to the loss of the hydrogen bond between it and Gln 92 (4.0 ). Interestingly, the shift seen in W1 and W2 in the proton transfer water network places the W2 water molecule at 3.9 distance from His64, suggesti ng a very weak hydrogen bond interaction (maximum hydrogen bond distance is often taken to be ~3.5 ). As an alternate proton transfer route, 230 240 HCA II seems to incorporate an additional water molecule in the active site (termed W2A; Figure 8 1B) that provides an alternative route between W2 and W3A from 3.4 to ~2.8

PAGE 114

114 . Proton transfer between W3A and His64 is then able to occur at distan ce similar to that seen in the native structure (3.3 ; Figure 8.4 ). 8.3.2 Thermal Stability Thermograms for the 230 240 HCA II variant were obtained in triplicate before being averaged, buffer subtracted and baseline corrected ( Figure 8 2). The major tra nsition peak for both variants displayed a single unfolding dynamic with a T M at ~59 C. The modest increase in thermal stability is encouraging, but further stability may be needed for biomedical and industrial use. 8.3.3 Kinetics The pH profiles for the two rate constants, k cat /K M and R H2O were determined by measuring the exchange of 18 O label between CO 2 and H 2 O via mass spectrometry. The rate of catalyzed interconversion between CO 2 and HCO 3 as measured by k cat /K M (Eq. 4 6; Table 8 2 ) for 230 240 HCA II ( Figure 8 3 A ) was decreased by two fold compared to wild type HCA II (66 versus 120 M 1 s 1 respectively). These two fold fluctuations, however, are not considered to be significant. Measurement of k B from Eq. 4 7 on Figure 8 3B revealed a proton transfer rate for 230 240 HCA II similar to that of wild type HCA II (Table 8 2). Inspection of the solved 230 240 HCA II crystallographic structure reveals a slightly elongated, but more linear, proton transfer water network when compared to th e native structure ( Figure 8 1B). The decreased catalytic efficiency of 230 240 HCA II may be associated with lowered active site pK a values as discussed in Section 7.3 .3 and/or to the increase in hydrogen bond interactions of the proton transfer water mo lecules.

PAGE 115

115 8.4 Discussion It has been postulated that thermophilic enzymes owe their characteristic stability to their relative rigidity compared to mesophilic homologues. Among the various examples for this hypothesis is the recently solved X ray crystallog raphic structure of an CA isolated from the hyperthermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) ( 203 ) which contains the loop deletion between residues 230 and 240 (HCA II numbering). Successful deletion of this loop region in the 230 240 HCA II variant was confirmed via X ray crystallography to a high resolution of 1.35 . Interestingly, this variant required a higher concentration (55 mg/mL) for crystallization than the wild type HCA II (10 mg/mL), and does so in the same crystal lization conditions but in different space groups (P2 1 2 1 2 1 versus P2 1 ). This is due to the 230 240 loop in wild type HCA II being a crystallographic contact point in the P2 1 crystallization conditions. Deletion of this loop removes the contact point, allow ing for closer packing of the enzyme within the unit cell and a higher symmetry. Thermal stability measurements via DSC, however, revealed only a ~2 C increase in the 230 240 HCA II variant as compared to the native enzyme (Table 8 2) The deletion of this loop region in SspCA cannot account for the remarkable thermal stability displayed by this enzyme, resisting denaturation for several hours in temperatures above 100 C ( 203 ) The authors do note, however, tha t another key difference between the structures of SspCA and HCA II is the high density of ionic pairs on the enzyme surface. Multiple salt bridge pairs along the surface have been also correlated to enhanced thermal stability in other proteins ( 188 )

PAGE 116

116 S urprisingly, the loop between residues 230 and 240 in HCA II may serve a role in catalysis as part of the extended active site, as evidenced by the two fold decrease in catalytic efficiency in the 230 240 HCA II variant as compared to wild type (Table 8 2 ). Visual inspection of the variant active site revealed a distorted proton transfer water network that included shifts in the positioning of W1, W2 W3B and W4, as well as the introduction of W2A into the active site ( Figure 8 1B). It is interesting to no te that coupled with the detrimental stretching of the W2 from His64 (3.9 ) is the rescue of the proton transfer rate via an alternative pathway that may include proton transfer from W3A to His64 (3.2 ) to the bulk solvent ( Figure 8 4) resulting in a k B value comparable to the native enzyme (Table 8 2). As discussed with the E234P HCA II variant in Section 7.4, the deletion of the acidic loop alters the pK a of the active site (as measured from Eq. 4 7 and Figure 8 3B ; Table 8 2 ), thereby providing a poss ible source for the lowered catalytic efficiency 8.5 Conclusions The idea of increasing the thermal stability of an enzyme by increasing its rigidity was investigated via deletion of the loop between residues 230 and 240 in HCA II. Exclusion of these 11 residues from the protein sequence resulted in a fully folded and functional enzyme that had a modest increase in thermal stability by ~2 C as compared to the full length wild type HCA II. This is a slightly disappointing increase in thermal stability pro vided that SspCA, which is kinetically active above 100 C for several hours, has the loop deletion as 230 may be accounted for by the abundance of salt bridge pairs along the surface of the enzyme. The catalytic efficiency of the 230 240 HCA II variant, however, was

PAGE 117

117 compromised by two fold compared to the native enzyme. This was highly unexpected as this loop region is sufficiently far from the Zn 2+ metal in the active site (>15 ) and is not involved in any interactions with the residues that are involved in maintaining the proton transfer water ne twork. Deletion of this highly acidic loop did seem to alter the pK a of the active site and could provide an explanation for the decreased kinetics. The large decrease in pK a of His64 measured in the 230 240 HCA II variant (Table 8 2) caused a reconfiguration of the water molecules in the active site as to not lose the characteristically high proton transfer rate ( Figure 8 4). This reconfiguration involves the crucial hydrogen bond interaction necessa ry for proton release into the bulk solvent occurring between W3A and His64 for 230 240 HCA II, as opposed to their being three potential routes for the proton to travel to His64 in the native enzyme ( Figure 8 1B). If this were correct, addition of the Y7 F mutation in the 230 240 HCA II variant should have a dramatic effect on catalytic efficiency by significantly lowering the proton transfer rate. Other possible loop truncat ion regions in HCA II include between residues 97 104 and 168 172. However, a s evidenced in Section 7.3.3 with the K170P HCA II variant, deletion of residues 168 172 may have detrimental effects on the catalytic efficiency of HCA II.

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118 Table 8 1. X ray crystallographic data set and refinement statistics for 230 240 HCA II Wavel ength () 0. 9177 Spacegroup P2 1 2 1 2 1 Unit cell parameters (;) a = 43.1 b = 69.0 c = 42.0 ; 90.0 Total number of measured reflections 168087 Total number of unique reflections 44710 Resolution () 20.0 1.35 (1.39 1.35)* R sym a (%) 12. 0 (48.0) I / (I) 11.2 (2.0 ) Completeness (%) 91.9 (93.8 ) Redundancy 3.8 (3.3 ) R work b (%) 20.8 R free c (%) 24.5 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensities. b R work o F c o |) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refinement (5% of reflections for all data sets). Values in parenthesis are for the highest resolution shell.

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119 Table 8 2. Thermal stability and catalytic m easurements for 230 240 HCA II Enzyme T M ( C) a k cat ex /K eff S (M 1 s 1 ) b k B 1 ) b pK a ZnOH b pK a His64 b wild type HCA II 5 7.1 ( 0 .1) 120 ( 20) 1.2 ( 0.2) 6.9 ( 0.1) 7.2 ( 0.1) 230 240 HCA II 58.9 ( 0.2 ) 66 ( 3) 1.1 ( 0.6) 6.7 ( 0.2) 5.8 ( 0.2) a Denaturation temperature as determined by DSC. b The rate c onstants are determined by fitting Eqs. (4 6) and ( 4 7) to Figures 8 3

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120 Figure 8 1. Crystallographic structure of 230 240 HCA II. A) Superimposition of 230 240 HCA II (pink) with the native enzyme (yellow). B) Active site water network configuration in 230 240 HCA II (red spheres) overlaid with the wild type enzyme water c oordinates (light pink spheres). Potential hydrogen bond interactions for the variant HCA II are shown as black dashed lines whereas the white dashed lines represent the hydrogen bonds seen in the native structure. Residues and water molecules are as label ed.

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121 Figure 8 2. Thermogram for 230 240 HCA II. Experimental data is shown as a black line with the fit to the data shown in red.

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122 Figure 8 3. Catalytic activity of 230 240 HCA II. A) Catalytic efficiency fit assuming a double ionization constant (closed circles) and B) pH profile o f 230 240 HCA II showing the measured rates for R1/[E] (closed circles) and R H2O /[E] (open squares).

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123 Figure 8 4 Schematic of the proton transfer water n etwork A) wild type HCA II and B) 230 240 HCA II. Hydrogen bond interactions are depicted as arr ows with the distance between atoms label in

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124 CHAPTER 9 COMBINATION OF STABILIZATION ELEMENTS 9.1 Overview The previous chapters have investigated the biophysical effects of several proposed thermal stabilizing elements in HCA II on the global structure melting temperature and catalytic efficiency of the enzyme. The ideal HCA II variant for industrial carbon sequestration protocol (Chapter 2) would require catalytic activity up to 70 C in a slightly acidic pH (<6.0). Additionally, the prime variant can didate for these processes must retain the characteristic high catalytic efficiency seen with wild type HCA II. As such, the nonpolar to polar surface mutations (TS1 HCA II ; Chapter 4) will be combined with the disulfide linkage between residues 23 and 203 (DS1 HCA II ; Chapter 5) for enhanced thermal stability. These two series of mutations are also beneficial in that they do not have any detrimental effect on the kinetics of HCA II The Y7F mutation will also be included with the surface mutations (TS2 HCA II; Chapter 4 ) and disulfide bridge formation (DS1 HCA II; Chapter 5 ) in an attempt to create a highly thermal stable HCA II variant with a high rate of proton transfer. Presented in this chapter are the X ray crystallographic structures of these two vari ants, along with their thermal unfolding curves at pH 7.8 and 5.6, as well as the kinetic profiles for the two variants as a function of pH and temperature. 9.2 Methodology 9.2.1 Protein Expression and Purification The plasmid cDNA containing either the TS 1 (L100H/L224S/L240P) or TS2 (Y7F/L100H/L224S/L240P) with DS1 (A23C/L203C/C206S) was prepared using primers ordered from Invitrogen The cDNA was transformed into E coli XL 1 Blue

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125 supercompetent cells, expressed with E. coli BL21(DE3)pLysS cells and purif ied on an affinity column as described in Section 4.2.1. Proper disulfide formation was promoted via the addition of ~0.1 mM oxidized glutathione (final concentration) to the protein sample followed by overnight dialysis in 50 mM Tris HCl, pH 7.8. Oligomer ic complexes were excluded from the sample via a Superdex 75 gel filtration column and were visualized on a native gel (data not shown). 9.2.2 X ray Crystallography C rystal tray s were prepared for the TS1/DS1 and TS2/DS1 HCA II variants with a 1:1 ratio of the reservoir solution and the protein sample ( 50 mg/mL) in 5 L drops The reservoir solutions contained 500 L of 0.2 M MgCl 2 0.1 M Tris HCl pH 8.5, 3 0% ( w /v) PEG 4000. To promote favorable crystal growth, 1 L of ~100 mM bile acid inhibitor cholate (See APPENDIX A) in 50 mM Tris, pH 7.8 was added to the 5 L drop The crystals formed in approximately three months via the hanging drop vapour diffusion method. Diffraction data sets for the TS1/DS1 and TS2/DS1 HCA II variants were collected on an in house Rigaku R Axis IV ++ image plate detector with a RU H3R rotatin g Cu anode (K = 1.5418 ) operating at 50 kV and 22 mA. The X rays were focused using Osmic optics, followed by a helium purged beam path. The crystal to detector distance w as 130 mm for TS1/DS1 HCA II and 100 mm for TS2/DS1 HCA II Each image was collected for five minutes with 1 oscillations HKL2000 ( 193 ) was used to integrate, merge and scale the d ata sets. The variants crystallized in the orthorhombic space group P2 1 2 1 2 1 and diffracted to resolutions of 1.85 and 2.15 for TS1/DS1 and TS2/DS1 HCA II respectively. The diffraction and refinement statistics for both variants are summarized in Table 9 1.

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126 Initial phases of the variants were calculated using molecular replacement using the wild type HCA II structure (PDB: 3KS3; ( 68 ) ) with 5% of the reflections set aside for R free calculations. PHENIX.REFINE ( 194 ) was used in cycles of restrained refinement of the molecular model, alternating with manual building using COOT ( 195 ) Initial F o F c difference maps revealed negative density around the m utation sites, the engineered disulfide linkage and the weak CA inhibitor cholate Subsequent refinements showed excellent electron density in the 2F o F c maps for these respective sites The final R cryst and R free values are shown in Table 9 1. MOLPROBITY ( 216 ) was used to assess the quality of the final model. All structural figures were created in PyMOL ( 205 ) 9.2.3 Differential Scanning Calorimetry Thermograms for the TS1/DS1 and TS2/DS1 HCA II variants were collected in triplicate at pH 7.8 and 5.6 before being buffer substracted and baseline corrected. The DSC thermograms were analyzed as outlined in Section 4.2.3. 9.2.4 Catalytic Activity The pH profiles for TS1/DS1 and TS2/DS1 HCA II were collected at 25 C as discussed in Section 4.2.4. The enzymatic activity was also studied at a temperature range from 10 C to 70 C in 100 mM HEPES and 10 mM substrate at pH 7.6. After the reaction was equilibriated to each temperature, a small sample of enzyme (~0.2% v/ v) was added to the reaction vessel. Measurements of 18 O content of CO 2 were made over the following one to five minutes. The kinetic constants and ionization constants shown in Eqs. 4 6 and 4 7 (Section 4.2.4) were determined through nonlinear least squar es methods (Enzfitter, Biosoft).

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127 9.3 Results 9.3.1 X ray Crystallography The TS1/DS1 and TS2/DS1 HCA II variants crystallized in the orthorhombic P2 1 2 1 2 1 space group with unit cell dimensions of a = 4 2 b = 73 and c = 74 ( = 90 ) The TS1/DS1 and TS2/DS1 HCA II variants diffracted to resolutions of 1.85 and 2.15 respectively. The final R cryst /R free values for TS1/DS1 are 18.6 and 22.9 % respectively whereas the R work /R free values for the TS2/DS1 HCA II variant are 17. 3 and 23.3%, respectively. A summary of the collected diffraction and final refinement statistics is shown in Table 9 1. The initial F o F c maps following molecular replacement confirmed the successful mutation of the TS1/TS2 and DS1 sites, with the final omission map (Figs. 9 1A and B) Subsequent refinement and placement of the Zn 2+ metal ion in the active site and te bound in the active site of these HCA II variants (Figs. 9 3C and D) The cholate ligand is found in 1.0 occupancy for both structures with comparable B factors to the surrounding water molecules (~20 2 ). Some deviation is seen in the binding patterns of cholate to the TS/DS1 HCA II variants when compared to the wild type enzyme (Appendix A) such as being in a less strenuous configuration and with the inclusion of less and better ordered water molecules The hydroxyl groups of the cholate ligand are a ll ~2.7 3.0 to W3B, W4 and W5 ( Figure 9 1C). As seen with the wild type crystallographic structure (Appendix A; Figure A 1C), W1 and W2 in the proton transfer network have been evacuated from the TS/DS1 active sites upon binding cholate ( Figure 9 1C a nd D) leaving W2, W3A and W3B interacting with their respective hydrogen bond partners The TS2/DS1 HCA II cholate bound structure

PAGE 128

128 ( Figure 9 1D) revealed a similar binding pattern to TS1/DS1 HCA II ( Figure 9 1C) except in that W3A has been evacuated from the active site with introduction of the Y7F mutation This is consistent from the results seen in Section 4.3.1 with the TS1 and TS2 HCA II variant s and with Y7F in previous studies ( 32 ) The spacegroup in which the TS/DS1 HCA II variants crystallize (P2 1 2 1 2 1 ) has a more open active site compared to the wild type condition (P2 1 ), thereby lessening the effects of crystal packing domains on the binding patterns of chol ate (See Figure A 1 D ). It is also interesting to note that cholate cannot co crystallize with wild type HCA II in the P2 1 spacegroup (i.e., the crystallographic structure was determined via soaking of the drug into preformed wild type crystals). The TS/DS1 HCA II variants actually required the presence of cholate to promote favorable crystal formation in the P2 1 2 1 2 1 group. 9.3.2 Thermal Stability The thermograms for the TS1/DS1 and TS2/DS1 HCA II variants were obtained in triplicate at pH 7.8 and 5.6 ( Figu re 9 2) The denaturation profiles for TS1/DS1 HCA II at pH 7.8 ( Figure 9 2A) are similar to those seen for the DS1 HCA II variant ( Figure 5 2A), displaying the characteristic profile for multiple unfolding intermediate states. The transition state contain ing the highest change in specific heat (C p ) has a melting temperature (T M ) of ~77 C, whereas the secondary transition peak occurs ~71 C (Table 9 2). This is a remarkable increase in thermal stability when compared to wild type HCA II ( T M > 20 C). Upon placement in a slightly acidic environment (pH = 5.6), TS1/DS1 HCA II is seen to retain its stability with the major unfolding transition occurring at ~6 8 C ( Figure 9 2B). Unlike the DS1 HCA II variant which underwent a single unfolding transition at aci dic pH ( Figure 5 2B), the TS1/DS1 HCA II variant

PAGE 129

129 retains the characteristic profile for multiple unfolding intermediates at T M 6 0 C with the possible addition of a less energetic transition at ~51 C ( Figure 9 2B; Table 9 2). This is an even greater increase in the thermal stability of the TS2/DS1 variant in an acidic environment compared to native HCA II ( T M > 22 C; Table 9 2). The thermograms obtained for the TS2/DS1 HCA II variant at pH 7.8 ( Figure 9 2C) were comparable to TS1/DS1 HCA II in that there is evidence for multiple unfolding transition states. However, the T M of the intermediate states of TS2/DS1 HCA II are much closer together than those seen in TS1/DS1 HCA II ( Figure 9 2A) and occur ~6 C less (T M 71 C). The TS2/DS1 HCA II variant is, however, ~14 C more thermal stable than wild type HCA II at pH 7.8 (Table 9 2). The multiple unfolding intermediate states are retained for TS2/DS1 HCA II at pH 5.6 ( Figure 9 2D) with a T M M of TS2/ M o that seen with TS1/DS1 HCA II (Table 9 2). Interestingly, the TS2/DS1 HCA II variant is seen to be ~16 C more thermal stable than wild type HCA II at acidic pH, which correlates well to the incre ase in stability seen with the TS1/DS1 HCA II variant (Table 9 2). 9.3.3 Catalytic Activity The pH profiles for the two rate constants, k cat /K M and R H2O were determined by measuring the exchange of 18 O label between CO 2 and H 2 O via mass spectrometry. The rate of catalyzed interconversion between CO 2 and HCO 3 as measured by k cat /K M (Eq. 4 6; Table 9 2 ) for TS1/DS1 ( Figure 9 3A) and TS2/DS1 HCA II ( Figure 9 3C) was decreased by ~two fold compared to wild type HCA II (~40 versus 100 M 1 s 1 respectively). These two fold fluctuations, however, are not considered to be significant. Measurement of k B from Eq. 4 7 on Figure 9 3B and D revealed a n increased proton

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130 transfer rate for TS1/DS1 HCA II of ~4 and ~7 s 1 for TS2/DS1 HCA II, respect ively (Table 9 2). These approximate four and seven fold increase in k B could associated with decreased pK a of His64 (from ~7 in wild type HCA II to less than 6 for the TS/DS1 HCA II variants; Table 9 2), thereby promoting deprotonation as discussed with t he K170P and E234P HCA II variants (Section 7.3.3) as well as the 230 240 HCA II variant (Section 8.3.3). 9.4 Discussion The combination of previously determined thermal stabilizing elements that increase the hydrophilicity of the HCA II surface (Chapter 4) and a disulfide linkage between residues 23 and 203 (Chapter 5 ) dramatically increased the thermal stability of the variant enzyme when compared to wild type HCA II by ~20 C at near physiological pH and >22 C in a more acidic environment (Table 9 2). The addition of the catalytically enhancing mutation Y7F in the a ctive site of the TS1/DS1 HCA II variant (TS2/DS1) resulted in less thermal stable enzyme when compared to a wild type active site of ~6 C, but was still more stable when compared to native HCA II by ~14 C. The loss in thermal stability has been observed before with the addition of the Y7F mutation (Table 4 2) and is associated with the loss of a hydrogen bond between the hydroxyl group of Tyr7 and W3A of the proton transfer water molecule in the active site ( Figure 4 1C). Interestingly, a decrease in T M of ~6 C in seen in Y7F HCA II at pH 7.8 when compared to the wild type HCA II (Table 4 2) identical to that seen when comparing TS1/DS1 and TS2/DS1 (Table 9 2) This suggests that native HCA II may have evolved to contain a Tyr residue at position seven for favorable N terminus stabilization with a well ordered water molecule that also limits the rate of proton transfer during catalysis. Comparison

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131 of the melting temperatures for the TS1, TS2 and DS1 HCA I I variants ( Figure 4 2B,C and 5 2 respectively) t o those of TS1/DS1 and TS2/DS1 ( Figure 9 2) variants suggest that the combination of these stabilizing elements has a linear effect. That is, the increase in thermal stability of the TS1 mutations compared to wild type HCA II ( T M 7 C) is additive to the increase seen with the DS1 mutation ( T M 14 C) so that the TS1/DS1 HCA II variant has a T M 20 C at pH 7.8. Interestingly, the addition of the Y7F mutation to the TS1/DS1 background variant resulted in a larger decrease in the melting temperature ( T M 6 C) than comparing the TS1 and TS2 HCA II variants ( T M 4 C; Table 4 2). The increase in thermal stability of the combination variants (TS1/DS1 and TS2/DS1) at acidic pH, however, were greater than those previously s een with the individual group of stabilizing mutations TS1, TS2 (Table 4 2) and DS1 (Table 5 2) as both the combination variants had an additional 2 C increase in T M when compared to the individual variants. The catalytic activity of the TS/DS1 HCA II va riants as measured by 18 O mass spectrometry revealed an insignificant two fold decrease in the catalytic efficiency compared to the wild type enzyme but with vastly enhanced proton transfer rates from four to seven fold higher than native HCA II (Table 9 2). The modest decrease in catalytic efficiencies of the TS/DS1 HCA II variants are surprising when considering that the TS1, TS2 and DS1 HCA II variants all displayed catalytic activity almost identical to the wild type enzyme (Tables 4 2 and 5 2). Simila rly, the increase in proton transfer of the TS1/DS1 HCA II variant (Table 9 2) is unexpected as both TS1 and DS1 HCA II displayed similar rates to wild type HCA II (Tables 4 2 and 5 2) The TS2/DS1 HCA II variant, however, displayed a proton transfer rate comparable to both the TS2 and Y7F

PAGE 132

132 HCA II variants (Table 4 2) As the X ray crystallographic structure of the TS/DS1 HCA II variants required the ligation of cholate, and thus the evacuation of ZnOH and W1 from the active site, the proton transfer water network in these variants remains unambiguous. However, the positioning of W2, W3A and W3B in the TS/DS1 HCA II variants are identical to those seen in the wild type active site, both in the presence and absence of the cholate ligand (See Figure A 1). As s uch, the most likely source for the altered catalytic activities of the TS/DS1 HCA II variants may lie in the lowered pK a values of His64 compared to the wild type enzyme (Table 9 2). The X ray crystallographic structures of the TS1/DS1 and TS2/DS1 HCA II variants ( Figure 9 1) required high concentrations of protein (50 mg/mL) and the addition of a hormonal inhibitor of HCAs, the bile acid cholate (Appendix A) in crystallization conditions comparable to those found with the TS HCA II variants (orthorhombic P2 1 2 1 2 1 spacegroup) and not those seen with the wild type enzyme and DS1 variant (monoclinic P2 1 spacegroup). Further inspection into the crystallographic packing of the symmetry related molecules of the combination HCA II variants revealed that the incre ased hydrophilicity surface is a new contact point in the orthorhombic spacegroup. Additionally, a secondary coordination site for a Mg 2+ metal ion (present from the crystallization conditions) is located along a crystallographic contact point that include s His4 and His64 from the asymmetric unit molecule and His36 of the symmetry related molecule. The B factor of the Mg 2+ ion is comparable to that seen with the Zn 2+ in the active site (~10 2 ) and provides an additional binding site for a highly dynam ic ch olate molecule (B factor ~5 0 2 ; max electron density ~0.3 e/A 3 ). The formation of /DS1 HCA II variant s is purely a consequence of

PAGE 133

133 crystal formation and not biologically significant, but is of scientific curiosity. Co mparison of the cholate bound structures in TS1 and TS2/DS1 HCA II to the wild type structure ( Figure A 1A) reveals the artificial crow d ing of molecules around the opening of the active site has an effect on the configuration of cholate bound in the respec tive active sites. The orthorhombic packing structures reveal a more relaxed conformation for cholate than seen in the monoclinic wild type structure. The strain imposed on cholate as a result from the crystallographic packing in the monoclinic spacegroup is further emphasized, as all attempts to co crystallize HCA II with cholate in this spacegroup have failed but happen readily in the orthorhombic spacegroup. These results indicate that crystallographic restraints created from contact points may significa ntly influence the successful formation of some protein/ligand complexes and require alternative crystal formation conditions and strategies. 9.5 Conclusions These investigations emphasize the ability to fine tune the biophysical characteristics of HCA I I including the thermal stability and catalytic activity with moderate predictability Expanding upon the thermal stabilizing elements presented in Chapters 4 and 5, the thermal and acid stability of HCA II was greatly increased via the combination of thes e mutations in the TS1/DS1 variant. Additionally, these stabilizing background mutations can be used in conjunction with the previously determined kinetically enhancing Y7F HCA II variant to produce a highly stable and active enzyme. Both th e TS 1 /DS1 and T S 2 /DS1 HCA II variants would be prime candidates for biomedical and industrial use as they display a n increased melting temperature (in excess of 70 C) and catalytic rates (Table 9 2), are highly soluble (>150 mg/mL) and

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134 are easily purified in massive qua ntities. The futility of TS1/DS1 HCA II in an artificial lung system is currently in progress in collaboration with Dr. William Federspiel in the McGowan Institute for Regenerative Medicine at the University of Pittsburgh with encouraging results. T he TS1/ DS1 and TS2/DS1 HCA II variants can be further used as a background for other stabilizing and/or kinetically enhancing mutations such as the Aros (Chapter 6) and/or N67Q (Chapter 4) HCA II variants.

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135 Table 9 1. X ray crystallographic data set and refinem ent statistics for TS1/DS1 and TS2/DS1 HCA II TS1/DS1 TS2/DS1 Wavelength () 1.5418 1.5418 Spacegroup P2 1 2 1 2 1 P2 1 2 1 2 1 Unit cell parameters (;) a = 41.8 b = 72.6 c = 74.3 ; 90.0 a = 42.0 b = 72.8 c = 74.5 ; 90.0 Total number of measured re flections 159611 114736 Total number of unique reflections 19081 12891 Resolution () 20.0 1.85 (1.92 1.85)* 20.0 2.15 (2.23 2.15) R sym a (%) 5.7 (41.4) 11.9 (49.8) I / (I) 30.6 (4.8 ) 15.4 (4.0) Completeness (%) 95.7 (92.8 ) 100.0 (100.0) Red undancy 8.4 (8.1 ) 8.7 (8.9) R cryst b (%) 18.6 17.3 R free c (%) 22.9 23.3 Number of residues 259 Number of protein atoms (including alternate conformations) 2256 Number of water molecules 127 r.m.s.d.: Bond lengths (), angles () 0.011, 1.400 Rama chandran statistics (%): Most favored, additionally allowed, generously allowed and disallowed regions 87.5, 11.1, 1.4 0.0 Average B factors ( 2 ): All, main chain, side chain, ligand, solvent 11.2, 9.0, 13.1 15.5, 19.8 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensities. b R cryst o F c o | ) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refinement (5% of reflections for all data sets). Values in parenthesis are for the highest resolution shell.

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136 Table 9 2. Thermal stability and catalytic m easure ments of the TS1/DS1 and TS2/DS1 HCA II variants Enzyme T M ( C) a k cat ex /K eff S (M 1 s 1 ) b k B 1 ) b pK a ZnOH b pK a His64 b pH 7.8 pH 5.6 wild type HCA II 57.1 ( 0.1) 45.4 ( 0.1) 100 ( 1 0) 1.2 ( 0.2) 6.9 ( 0.1) 7.2 ( 0.1) TS1/DS1 77.4 ( 0.1) 71.0 ( 0.2 ) 67.9 ( 0.1 ) 60.4 ( 0.2 ) 50.8 ( 0.1 ) 38 ( 3) 4.2 ( 0.8) 7.4 ( 0.1) 5.9 ( 0.1) TS2/DS1 71.2 ( 0.1 ) 61.0 ( 0.1 ) 57.5 ( 0.4 ) 44 ( 4) 6.8 ( 1.4) 6.7 ( 0.1) 5.7 ( 0.1) a Denaturation temperature as determined by DSC. b The rate c onstants are determined by fitting Eqs. (4 6) and ( 4 7) to the data of Figure 9 3

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137 Figu re 9 1. Global structure of the TS1/DS1 and TS2/DS1 HCA II variants. Final 2F o F c electron density maps contoured at of the TS1/DS1 variant for the A) L100H, L224S and L240P mutations and B) A23C/L203C disulfide linkage. C) Binding interactions of cholate to TS1/DS1 and D) TS2/DS1 HCA II. Residues and waters are as labeled with hydrogen bonds indicated a s black dashed lines.

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138 Figure 9 2. Thermograms of TS1/DS1 and TS2/DS1 HCA II at pH 7.8 and 5.6. The major unfolding transitions for TS1/DS1 at pH A) 7.8 and B) 5.6. The thermograms for TS2/DS1 at pH C) 7.8 and D) 5.6 are shown. Experimental data is show n in black with the fit to the data shown in red.

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139 Figure 9 3. Catalytic activity of the TS1/DS1 and TS2/DS1 HCA II variants. A) Catalytic efficiency of the TS1/DS1 HCA II variant (closed circles) and B) pH profile showing the rates R 1 /[E] as closed ci rcles and R H2O /[E] as open squares. The same are shown in C) and D) but for TS2/DS1 HCA II. E) The rate constant R 1 /[E] (s 1 ) as a function of temperature for the interconversion of CO 2 and bicarbonate catalyzed by variants of HCA II : w ild type HCA II bl ack squares; TS1/DS1 HCA II: red diamonds; TS2/DS1 HCA II : blue triangles.

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140 CHAPTER 10 CONCLUSIONS Bioengineering of enzymes to meet specific criteria can be a very powerful technique. With the ever increasing world demand for climate change and need for biomedical catalysts, it is essential for the rational design of thermal stable variant of HCA II without any detrimental effects to its characteristically high catalytic activity. Through comparison of thermophilic and mesophilic enzymes, the blueprints for the bioengineering of thermal stable HCA II variant was constructed. Basic principles included lowering the surface hydrophobicity and establishing new hydrogen bonds (both intra and inter molecular), establishing covalent disulfide linkages, incorpor ating an aromatic cluster into the hydrophobic core of the enzyme and loss of surface malleability via either increasing loop rigidi ty through proline substitution or deletion of entire loop segments. The stabilization of these elements on HCA II was analy zed via DSC with confirmation of the catalytic activity of the enzyme utilizing 18 O mass spectrometry. The underlying biophysical principles as to the stabilization/destabilization effects these various elements have on HCA II were visualized using X ray c rystallography. Of the proposed stabilizing elements, the greatest thermal stabilization of the HCA II variants involved the incorporation of an additional aromatic cluster with the mutations L47F/V49F/I146F/L212F (Chapter 6 ), with an increase of over 25 C in melting temperature. This correlates well with protein folding theory which states that the hydrophobic core of a protein is the initiating step of protein folding, and is primarily driven from the hydrophobic effect of increasing the entropy of the s ystem upon folding.

PAGE 141

141 This increase in stability was also associated with unfortunately, a threefold decrease in the catalytic activity of this HCA II variant. Other proposed entropic stabilization mechanisms including lowering of HCA II surface loop entrop y via proline point mutations (Chapter 7) or entire loop deletions (Chapter 9) resulted in minor thermal stabilization (~2 C) at best but also adversely affected the catalytic efficiencies by two fold. However, successful reduction of the surface entropy of HCA II (and stabilization of the folded stated) can be achieved via proline substitution if the mutated residue is more hydrophobic than proline as evidenced by the L240P variant (Chapter 4). The loss in catalytic efficiencies in the K170P, E234P (Chapt er 7) and 230 24 0 HCA II variants suggests that residues that extend around the peripheral of the active site play an essential role in the catalytic mechanism of HCA II, possibly serving as a pK a regulator for the active site. Increasing the enthalpic contribution s along the HCA II surface via mutation of nonpolar leucines into polar residues had a significant effect of the thermal stability of the variants (Chapter 4). Interestingly, these newly established hydrogen bonds not only provided stability via intramolec ular interactions within the protein but also intermolecular interactions were created with the influx of solvent molecules around the mutation sites. Additionally, the loss of a hydrogen bond with a well ordered water molecule in the active site of the Y7 F HCA II variant destabilizes the enzyme while at the same time increasing the proton transfer rate (Chapter 4). With the insight that the addition of a few weak hydrogen bond interactions add significantly to the thermal stability of HCA II, establishing new covalent disulfide bridges should further enhance thermal resistance. As outlined in Chapter 5, disulfide linkages can significantly increase

PAGE 142

142 the thermal stability by up to 14 C, but the positioning of the bridge greatly affects the stabilization and catalytic activity of HCA II. The best strategy for engineering of disulfide bridges may be through superimposition of homologues containing naturally occurring linkages. The most promising thermal stabilizing elements could additionally be combined togeth er into highly stabilized HCA II variants with increased catalytic activity as outlined in Chapter 9. These elements included reduction of the surface hydrophobicity and construction of a disulfide bridge between residues 23 and 203 in HCA II. This variant of HCA II (TS1/DS1) is ~20 C more stable than the wild type enzyme at near physiological and acidic pHs with comparable catalytic efficiency. The inclusion of the proton transfer rate enhancing mutation Y7F (TS2/DS1) yielded a HCA II variant with a ~14 C increase in thermal stability and enhanced catalytic rates. These variants are outstanding candidates to be used in industrial and biomedical applications such as in bioremediation processes and implementation in artificial lung systems.

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143 APPENDIX HORM ONAL INHIBITION OF HCA II VIA THE BILE ACID CHOLATE A.1 Overview Several isoforms of HCAs along the gastro intestinal tract, including HCA II, are inhibited by the bile acids (BAs) ( 220 ) the primary products of cholesterol catabolism ( 221 ) BAs have been shown to damage the gastric mucosa in animals, and duodenogastric reflux is regarded as one pathogenic factor in gastritis, gastric ulcer and alkaline reflux gastritis in the postgastrectomy stomach in humans ( 222 ) The mucosal damaging activity of cholic acid, taurocholic acid and glycochenodeoxycholic acid has been directly associated with gastric mucosal CA inhibition in rats and humans ( 223 ) The bile acids have been shown to be transported into the cytosol of bil iary epithelial cells ( 224 ) The immediate products of the BA synthetic pathways (cholic acid and chenodeoxycholic acid) are referred to as primary BAs, whereas dehyroxylation from C7 of the sterane core of either primary BA via the action of the gut bacterial flora constitutes the secondary BAs, deoxyc holic and lithocholic acid The hydrophilicity of the BAs can be further enhanced before secretion into the bile canalicular lumen of the gall bladder for storage via conjugation with taurine or glycine ( 221 ) The strongest binding affinity among the BAs for HCA II include c holic and deoxycholic acid, with I 50 values of ~0.1 and 0.4 mM, respectively, as measured via 18 O mass spectrometry ( Figure A 2) and previous stopped flow flow spectrophotometry studies ( 222 ) Structural knowledge of the binding interactions of the BAs to HCA II would promote further insights into gastric ulcer development and aid in the design of a therapeutic agent that displays isoform inhibition specificity among the HCAs. The inhibition of HCA II has been utilized for the treatment of various diseases in the past

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144 including glaucoma, epilepsy and altitude sickness ( 47 ) Current clinically used inhibitors of HCA II include acetazolamide and brinzolamide which incorporate a sulfonamide functional head group that have detrimental side effects including augmented diu resis, fatigue, paresthesias and anorexia due to the non specific inhibition of CA isoforms in other tissues than those targeted ( 16 ) Additionally, an estimated 3% of t he total population has an adverse drug reaction to sulfonamide containing compounds, with higher rates of sulfa allergies seen in individuals with low metabolic process rates and immuno compromised patients ( 225 ) Thus, BA inhibition of HCA II is an attractive therapeutic agent against glaucoma as they are non sulfur containing compounds, easily transported across the cellular membrane and are extremely soluble in water compared to conventional drugs such as aceta zolamide and methazolamide. A.2 Methodology A.2.1 Enzyme Expression and Purification HCA II cDNA containing the enzyme coding region ( 141 ) was transformed into Escherichia coli XL1 Blue super com petent cells, which were then confirmed by DNA sequencing of the entire coding region. The HCA II cDNA was then transformed in Escherichia coli BL21(DE3) cells in 1L of 2 x Luria broth medium containing ~0.1 mg/mL ampicillin and grown at 37 C to a turbidi ty of ~0.6 at 600 nm. Protein production was D 1 thiogalactopyranoside (IPTG) and ~1 mM zinc sulfate (final concentrations). The cells were incubated for an additional three hours and harvested by centrif ugation. A suspension of cells in 200 mM sodium sulfate, 100 mM Tris HCl, pH 9.0 was lysed by addition of hen egg white lysozyme and DNaseI with subsequent removal of

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145 cellular debris by centrifugation. The enzyme was purified on affinity column containing an agarose resin coupled with p (aminomethyl) benzene sulfonamide, a tight binding inhibitor of HCA II ( 192 ) The bound HCA II was eluted with 400 mM sodium azide, 100 mM Tris, pH 7.0 followed by extensive dialysis in 50 mM Tris HCl, pH 7.8 to remove the azide. The purified enzyme was concentrated to ~10 mg/mL for crystallization studies via centrifugal ultra filtration using a 10 kDa MWCO filter (Amicon). The purity of the sample was visualized via SDS PAGE and determined to be >95% (data not s hown). A.2.2 Crystallization and Diffraction Data Collection Crystals of HCA II were observed after one week in a 1:1 ratio of protein: reservoir citrate, 50 mM Tris HCl, pH 7.8. After crystal HCl, pH 7.8, was soaked into the crystal to a final concentration of ~17 mM for 4 hours prior to data collection. The crystal was further washed for 1 0 secs in cryo protectant containing an equivalent concentration of cholate dissolved in the mother liquor and 20% (w/v) glycerol prior to data collection. Diffraction data were collected at 100K on an in house Rigaku R Axis IV ++ image plate detector using an RU H3R rotating Cu anode (K = 1.5418 ) operating at 50kV, 22mA with a crystal to detector distance of 80 mm. The X rays were focused via Osmic optics, followed by a helium purged beam path. Diffraction data were collected with 1 oscillations and an exposure time of 300 seconds pe r image. HKL2000 ( 193 ) was used to integrate, merge, and scale the data in the monoclini c space group P2 1 to a final resolution of 1.54. A summary of the data statistics is provided in Table A 1

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146 Initial phases for the HCA II/cholate were calculated via molecular replacement using the coordinates of the high resolution, anisotropic HCA II st ructure (PDB: 3KS3) ( 68 ) Electron density in the initial F o F c suggest cholate binding in the active site of HCA II. PHENIX.REFINE ( 226 ) was used in cycles of restrained refinement of th e molecular model, alternating with manual building using COOT ( 227 ) Solvent molecules refined with B factors of more than 50 2 were excluded from the final model. The R cryst and R free valu es for the final model were 14.7% and 17.4 %, respectively. PROCHECK ( 204 ) was used to assess the quality of the final model. 88% of the residues were in most favored conformation, 11.5% generously allowed and 0.0% was in disallowed regions. The final model st atistics are included in Table A 1 All crystallograp hic figures were generated in PyMOL ( http://www.pymol.org) Experimental data and structural coordination have been deposited with the Protein Data Bank under the accession number 4N16. A.2.3 Inhibition Studies An 18 O exchange assay was carried out to stud y the kinetics of the catalyzed HCO 3 CO 2 dehydration hydration reaction and its bile acid inhibition at 283 K. This method relies on the depletion of 18 O from CO 2 as measured by membrane inlet mass spectrometry. CO 2 passing across the membrane of the in let enters a mass spectrometer (Extrel EXM 200), providing a continuous measure of the isotopic content of CO 2 in solution. In the first stage of catalysis, the dehydration of labeled bicarbonate has a probability of transiently labeling the active site wi th 18 O. In a subsequent step, protonation of the zinc bound 18 O labeled hydroxide results in the release of H 2 18 O into the solvent. I 50 values were obtained for cholate and deoxycholate in 100 mM HEPES (pH 8.0). Dissolved bile acids (100 mM) in 100 mM Tris (pH 7.8) was tit rated against

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147 18 O had occurred. A.3 Results The X ray crystallographic structure of cholate bound to HCA II was refined to a resolution of 1.54 with the fina l model ( Figure A 1A ) hav ing values for R cryst and R free of 14.7 and 17.4%, respectively. A summary of the collected diffraction sets and final refinemen t statistics is shown in Table A 1 Initial F o F c phase maps obtained via molecular replacement with the high resolution, anis otropic, model of unbound HCA II the active site. The final 2F o F c electron density map contoured at 1.3 surroun ding cholate is shown in Figure A 1B The observed va lues of I 50 were based on the measurement of the catalytic activity of HCA II by 18 O mass spectrometry and revealed weak binding affin ity for cholate (~0.2 mM; Figure A 2 A) an d deoxycholate (~0.4 mM; Figure A 2B ). These values agree well with inhibition da ta performed in previous stopped flow experiments (~0.1 and 0.4 mM, respectively) ( 223 ) The hydrox yl groups of cholate do not make any potential strong hydrogen bond interactions with the hydrophilic face of the HCA II active site. Instead, cholate is observed to utilize a series of water molecules that provides a weak connection betwe en the enzyme and ligand (Fig A 1C ). Cholate is seen to bind to the zinc ion in a bivalent interaction centralized between the two oxygens of the carboxyl group which displaces the zinc bound solvent molecule and Water1 (W1) of the proton transfer water network ( 1 ) The carboxyl oxygens are also in potential hydrogen bond distance with two well ordered water molecules seen in the unbound active site termed deep water (D W ), which acts as a place holder for CO 2 binding, and W2 which is involved in the

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148 transfer of a proton from the zinc bound solvent to His64 ( 1 ) The D W molecule has been displaced 0.9 further into the hydrophobic pocket of the CO 2 binding site towards Trp209 upon binding cholate whereas the zinc b ound solvent molecule whereas W2 is in an comparable position to the unbound structure. The branching water molecules involved in hydrogen bonding to Tyr7 and Asn62/67 (W3A and W3B, respectively) are also observed in the cholate bound structure. W2 and W3 A are seen in comparable positions as to those in the unbound structure, whereas W3B accompanies two distinguishable binding sites ~0.5 apart, termed W3B 1 and W3B 2 in ~60 and 40% occupancy, respectively. W3B 1 is within hydrogen bond distance of N 2 in Asn62 (3.0 ), which has shifted ~0.7 compared to the unbound structure. The lower occupancy W3B 2 molecule is within 2.6 of N 2 in Asn67, which has been flipped from its position in the unbound structure where W3B interacts with O 1 of Asn67. The relatively high B factor for O 1 of Asn67 (45 versus ~20 2 average) and weak electron density in the 2F o F c map (max peak at ~0.7 e/A 3 ) suggests dynamic hydrogen bond interactions between this carbonyl oxygen to N 2 of Asn62, N 2 of Gln92 and a low oc cupancy (<10%), diffuse water. Movement of N 2 of Gln92 ~1.6 compared to the unbound structure places it in position for a potential hydrogen bond to W6B (2.9 ) and a weak direct interaction to the C7 hydroxyl group of cholate (3.5 ). The C12 hydroxyl is seen to make potential hydrogen bond interactions with W5 (2.8 ), W6A and W6B (~2.8 ), which are present 1.7 from one another in ~50% occupancy each ( Figure 2C). W6A makes a potential hydrogen bond with N 2 in Asn62 (2.6 ) whereas W6B interacts wit h N 2 of Gln92 (above). The movements of W3A 1 toW3B 2 with W6A to W6B may be correlated as they are ~2.4 from one another

PAGE 149

149 with comparable occupancies. The thermal fluctuations seen in O 1 of Asn67 may be the driving force behind these alternate states of W3B and W6. W5 interacts with a neighboring water molecule (2.6 ) which is within hydrogen bonding distance of the carbonyl backbone of Pro201 (2.7 ). W5 and W2 are also bond to W4 which is within hydrogen bond distance of Thr200. W4 is commonly seen in other HCA II/inhibitor structures when His64 is locked into ( 228 ) The C7 hydroxyl is within hydrogen bonding distance with W7 (2.4 ), which binds to an adjacent water molecule that interacts with the backbone carbonyl oxygen of Ile91. A.4 Discussion The natural epimerization of the hydroxyl and methyl moieties on the sterane core presents an optimal configuration for cholate to interact with the opposing hydrophilic and hydrophobic faces found within t he active site of HCA II ( Figure A 1 C). The hydrophobic residues Ile91, Val121, Phe131, Leu141 and Leu198 are seen to have a significant buried surface area (>50%) upon cholate binding. Of these hydrophobic interfaces, Phe131 and Leu198 are seen to undergo the largest conformational shift (~0.7 0.8 ) upon ligation. Interestingly, Phe131 is highly variable among the human CA isozymes [i.e., Val in HCA IX ( 229 ) and Ala XII ( 230 ) two isoforms overexpressed on the outside of tumor cells. Utilization of t he slight hydrophobicity differences in the active site of these CAs overexpressed in an wide array of cancer lines could provide a site for isoform specificity. The C3 hydroxyl group is seen to be within hydrogen bon d distance of W8 (2.6 ; Figure A 1C white dashed line) which makes a hydrogen bond with Glu69 and W7. Closer inspection of the crystallographic structure revealed an ~90 tilt of ring A in the

PAGE 150

150 sterane core that is most likely due to close contact of residues 235 238 on a symmetry related mo lecule ( x+1, y 1/2, z+2) near the opening of the active site of the molecule in the asymmetric unit ( Figure A 1D ) and may not be biologically significant. A.4 Conclusions Crystallization of the conjugated BAs (e.g., glycocholate) with HCA II may be limi ted due to the steric interference of symmetry related molecules within t he monoclinic unit cell ( Figure A 1D ) and may require studies in spacegroups, such as P2 1 2 1 2 1 that increase the accessibility of the active site ( 93 ) Attempts to observe deoxycholate in the active site of HCA II were limited due to lowered solubility and higher critical micellular concentrations compared to cholate, but the weaker binding affin ity can be extrapolated as dehydroxylation from C7 would correspond to broken hydrogen bon ds between W5 and W6A/B ( Figure A 1C ). Implementation of substituents on the sterane core that establish direct hydrogen bonds among the active site residues such as Asn67 in HCA II (Gln67 in HCA IX) that could provide higher binding affinity and provide a template for isoform specificity among therapeutic cancer agents.

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151 Table A 1. X ray crystallographic data set and refinement statistics for HCA II/Cholate Waveleng th () 1.5418 Spacegroup P2 1 Unit cell parameters (;) a = 42.4 b = 41.6 c = 71.9 ; 104.5 Total number of measured reflections 1 24916 Total number of unique reflections 33813 Resolution () 20.0 1.54 (1.60 1.54 )* R sym a (%) 4.4 (44.1 ) I / (I) 26.9 (2.9 ) Completeness (%) 93.6 (89.8 ) Redundancy 3.7 (3.7 ) R cryst b (%) 1 4.5 R free c (%) 17.1 Number of residues 25 8 Number of protein atoms (including alternate conformations) 22 87 Number of water molecules 265 r.m.s.d.: Bond lengths (), angles () 0.009 1. 308 Ramachandran statistics (%): Most favored, additionally allowed, generously allowed and disallowed regions 88.0, 11.5, 0.5 0.0 Average B factors ( 2 ): All, main chain, side chain, ligand, solvent 13.4 9.0, 13.1 13.0 24.3 a R sym hkl i |(I i (hkl) is the average intensity for this reflection; the summation is over all intensities. b R cryst o F c o | ) x 100 c R free is calculated in the same way as R cryst except it is for data omitted from refinement (5% of reflections for all data sets). Values in parenthesis are f or the highest resolution shell.

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152 Figure A 1. X ray crystallographic structure of cholate binding to HCA II. (A) Surface view of cholate (orange sticks) binding into the active site of HCA II (transparent grey surface). The C backbone is shown in cartoon view in green. The Zn 2+ metal ion is shown as a magenta sphere. (B) Final 2F o F c II. (C) Hydrogen bond interactions (black dashed lines) between c holate and the surrounding water molecules (red spheres). Interacting residues and water molecules are as labelled. The water molecules important in the proton transfer mechanism (W2, W3A, W3B 1 and W3B 2 ) are shown as light pink spheres. (D) Crystal packing depicting the crystallographic symmetry molecule x+1, y 1/2, z+2 (cyan C cartoon).

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153 Figure A 2. Determination of I 50 from catalytic activity M easured by mass spectrometry using 18 O labeled substrates for (A) Cholate and (B) Deoxycholate. I 50 values are given inset.

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154 REFERENCES 1. Boone, C. D., Pina rd, M., McKenna, R., and Silverman, D. (2014) Catalytic Mechanism of a Class Carbonic Anhydrases: CO 2 Hydration and Proton Transfer, In Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications (Frost, S. C., and McKenna, R., Eds.), pp 31 52, Springer, New York. 2. Lindskog, S., and Coleman, J. E. (1973) Catalytic Mechanism of Carbonic Anhydrase, Proceedings of the National Academy of Sciences of the United States of America 70 2505 2508. 3. Lindskog, S., and Silverman, D. N. (2000) The catalytic mechanism of mammalian carbonic anhydrases, In The Carbonic Anhdyrases: New Horizons (Chegwidden, W. R., Carter, N. D., and Edwards, Y. H., Eds.), pp 175 195, Birkhuser Verlag, Boston, USA. 4. Krishnamurthy, V. M., Kaufman, G. K., Urbach, A. R., Gitlin, I., Gudiksen, K. L., Weibel, D. B., and Whitesides, G. M. (2008) Carbonic Anhydrase as a Model for Biophysical and Physical Organic Studies of Proteins and Protein Ligand Binding, Chem. Rev. 108 946 1051. 5. Price, G., and Badger, M. R. (1989) Ethoxyzolamide inhibition of CO 2 dependent photosynthesis in the Cyanobacterium Synechococcus PCC7942, Plant Physiol 89 44 50. 6. Kisker, C., Schindelin, H., Alber, B. E., Ferry, J. G., and Rees, D. C. (1996) A left hand B helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila EMBO J 15 2323 2330. 7. Parisi, G., Fornasari, M., and Echave, J. (2000) Evolutionary analysis of gamma carbonic anhydrase and structurally related proteins, Mol ecular phylogenetics and evolution 14 323 334. 8. Fu, X., Yu, L. J., Mao Teng, L., Wei, L., Wu, C., and Yun Feng, M. (2008) Evolution of structure in gamma class carbonic anhydrase and structurally related proteins, Molecular phylogenetics and evolution 47 211 220. 9. Lane, T. W., and Morel, F. M. (2000) Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii, Plant Physiol 123 345 352. 10. Sly, W. S., and Hu, P. Y. (1995) Human ca rbonic anhydrases and carbonic anhydrase deficiencies, Annu Rev Biochem 64 375 401.

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155 11. Frost, S. C. (2014) Physiological functions of the alpha class of carbonic anhydrases, In Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications (Frost, S. C., and McKenna, R., Eds.) 2013/10/23 ed., pp 9 30, Springer, New York. 12. Eriksson, A. E., Jones, T. A., and Liljas, A. (1988) Refined structure of human carbonic anhydrase II at 2.0 A resolution, Proteins 4 274 282. 13. Pocker Y., and Sarkanen, S. (1978) Adv Enzymol Relat Areas Mol Biol. 47 149. 14. Fisher, S. Z., Kovalevsky, A. Y., Mustyakimov, M., McKenna, R., Silverman, D. N., and Langan, P. (2010) The neutron structure of human carbonic anhydrase II: Implications for pro ton transfer, Biochemistry 49 415 421. 15. Christianson, D. W., and Fierke, C. A. (1996) Carbonic Anhydrase: Evolution of the Zinc Binding Site by Nature and by Design, Accounts of chemical research 29 331 339. 16. Aggarwal, M., Kondeti, B., and McKen na, R. (2013) Insights towards sulfonamide drug specificity in alpha carbonic anhydrases, Bioorganic & medicinal chemistry 21 1526 1533. 17. Aggarwal, M., and McKenna, R. (2012) Update on carbonic anhydrase inhibitors: a patent review (2008 2011), Expe rt opinion on therapeutic patents 22 903 915. 18. Alterio, V., Di Fiore, A., D'Ambrosio, K., Supuran, C. T., and De Simone, G. (2012) Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoform s?, Chem Rev 112 4421 4468. 19. Boriack, P. A., Christianson, D. W., Kingery Wood, J., and Whitesides, G. M. (1995) Secondary Interactions Significantly Removed from the Sulfonamide Binding Pocket of Carbonic Anhydrase II Influence Inhibitor Binding Cons tants, Journal of Medicinal Chemistry 38 2286 2291. 20. Aggarwal, M., Boone, C. D., Kondeti, B., and McKenna, R. (2013) Structural annotation of human carbonic anhydrases, J Enzyme Inhib Med Chem 28 267 277. 21. Hkansson, K., Carlsson, M., Svensson, L A., and Liljas, A. (1992) Structure of native and apo carbonic anhydrase II and structure of some of its anion ligand complexes, Journal of Molecular Biology 227 1192 1204. 22. Merz, K. M. (1991) Carbon dioxide binding to human carbonic anhydrase II, J ournal of the American Chemical Society 113 406 411.

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156 23. Tu, C. K., Silverman, D. N., Forsman, C., Jonsson, B. H., and Lindskog, S. (1989) Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site specific mutant, Biochemistry 28 7913 7918. 24. Steiner, H., Jonsson, B. H., and Lindskog, S. (1975) Catalytic Mechanism of Carbonic Anhydrase Hydrogen Isotope Effects on Kinetic Parameters of Human C Isoenzyme, European Journal of Biochemistry 59 253 259. 25. Nair, S. K., and Christianson, D. W. (1991) Unexpected pH Dependent Conformation of His 64, the Proton Shuttle of Carbonic Anhydrase II, Journal of the American Chemical Society 113 9455 9458. 26. Fisher, Z., Hernandez Prada, J. A., Tu, C., Duda, D., Yoshioka, C., An, H., Govindasamy, L., Silverman, D. N., and McKenna, R. (2005) Structural and kinetic characterization of active site histidine as a proton shuttle in catalysis by human carbonic anhydrase II, Biochemistry 44 1097 1105. 27. Shimahara, H., Yoshida T., Shibata, Y., Shimizu, M., Kyogoku, Y., Sakiyama, F., Nakazawa, T., Tate, S., Ohki, S. Y., Kato, T., Moriyama, H., Kishida, K., Tano, Y., Ohkubo, T., and Kobayashi, Y. (2007) Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase, The Journal of biological chemistry 282 9646 9656. 28. Nair, S. K., Ludwig, P. A., and Christianson, D. W. (1994) Two Site Binding of Phenol in the Active Site of Human Carbonic Anhydrase II: Structural Implications for Substrate Asso ciation, Journal of the American Chemical Society 116 3659 3660. 29. Fisher, Z., Kovalevsky, A. Y., Mustyakimov, M., Silverman, D. N., McKenna, R., and Langan, P. (2011) Neutron structure of human carbonic anhydrase II: a hydrogen bonded water network "s witch" is observed between pH 7.8 and 10.0, Biochemistry 50 9421 9423. 30. Nair, S. K., and Christianson, D. W. (1991) Unexpected pH dependent conformation of His 64, the proton shuttle of carbonic anhydrase II, Journal of the American Chemical Society 1 13 9455 9458. 31. Fisher, S. Z., Maupin, C. M., Budayova Spano, M., Govindasamy, L., Tu, C., Agbandje McKenna, M., Silverman, D. N., Voth, G. A., and McKenna, R. (2007) Atomic crystal and molecular dynamics simulation structures of human carbonic anhydra se II: insights into the proton transfer mechanism, Biochemistry 46 2930 2937.

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157 32. Fisher, S. Z., Tu, C., Bhatt, D., Govindasamy, L., Agbandje McKenna, M., McKenna, R., and Silverman, D. N. (2007) Speeding up proton transfer in a fast enzyme: kinetic a nd crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II, Biochemistry 46 3803 3813. 33. Mikulski, R., Avvaru, B. S., Tu, C., Case, N., McKenna, R., and Silverman, D. N. (2011) Ki netic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II, Archives of biochemistry and biophysics 506 181 187. 34. Mikulski, R., West, D., Sippel, K. H., Avvaru, B. S., Aggarwal, M., Tu, C., McKenna, R., and Silverman, D. N. (2013) Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II, Biochemistry 52 125 131. 35. Mikulski, R., Domsic, J. F., Ling, G., Tu, C., Robbins, A. H., Silverman, D. N., and McKenna, R. (2011) S tructure and catalysis by carbonic anhydrase II: role of active site tryptophan 5, Archives of biochemistry and biophysics 516 97 102. 36. Mikulski, R. L., and Silverman, D. N. (2010) Proton transfer in catalysis and the role of proton shuttles in carbon ic anhydrase, Biochimica et biophysica acta 1804 422 426. 37. Domsic, J. F., Williams, W., Fisher, S. Z., Tu, C., Agbandje McKenna, M., Silverman, D. N., and McKenna, R. (2010) Structural and kinetic study of the extended active site for proton transfer in human carbonic anhydrase II, Biochemistry 49 6394 6399. 38. Silverman, D. N., and Lindskog, S. (1988) The catalytic mechanism of carbonic anhydrase: implications of a rate limiting protolysis of water, Accounts of chemical research 21 30 36. 39. Kha lifah, R. G. (1971) The carbon dioxide hydration activity of carbonic anhydrase I. Stop flow kinetic studies on the native human isoenzyme B and C, J. Biol. Chem. 246 2561 2573. 40. Silverman, D. N., and McKenna, R. (2007) Solvent mediated proton transfe r in catalysis by carbonic anhydrase, Accounts of chemical research 40 669 675. 41. Eriksson, A. E., Kylsten, P. M., Jones, T. A., and Liljas, A. (1968) Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinate d binding of the SCN ion to the zinc at high pH, Proteins 4 283 293.

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158 42. Lindahl, M., Svensson, L. A., and Liljas, A. (1993) Metal poison inhibition of carbonic anhydrase, Proteins 15 177 182. 43. Williams, T. J., and Henkens, R. W. (1985) Dynamic carb on 13 NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I, Biochemistry 24 2459 2462. 44. Bertini, I., Luchinat, C., Monnanni, R., Roelens, S., and Moratal, J. M. (1987) Interaction of carbon dioxide and cop per(II) carbonic anhydrase, Journal of the American Chemical Society 109 7855 7856. 45. Krebs, J. F., Rana, F., Dluhy, R. A., and Fierke, C. A. (1993) Kinetic and spectroscopic studies of hydrophilic amino acid substitutions in the hydrophobic pocket of human carbonic anhydrase II, Biochemistry 32 4496 4505. 46. Supuran, C. T. (2008) Carbonic anhydrases -an overview, Current pharmaceutical design 14 603 614. 47. Supuran, C. T. (2008) Carbonic anhydrases: novel therapeutic applications for inhibitors a nd activators, Nature reviews. Drug discovery 7 168 181. 48. Supuran, C. T. (2010) Carbonic anhydrase inhibitors, Bioorganic & medicinal chemistry letters 20 3467 3474. 49. Supuran, C. T. (2012) Inhibition of carbonic anhydrase IX as a novel anticancer mechanism, World J Clin Oncol. 3 98 103. 50. Supuran, C. T., Scozzafava, A., and Casini, A. (2003) Carbonic anhydrase inhibitors, Medicinal research reviews 23 146 189. 51. Supuran, C. T., and Scozzafava, A. (2007) Carbonic anhydrases as targets for m edicinal chemistry, Bioorganic & medicinal chemistry 15 4336 4350. 52. Domsic, J. F., and McKenna, R. (2010) Sequestration of carbon dioxide by the hydrophobic pocket of the carbonic anhydrases, Biochimica et biophysica acta 1804 326 331. 53. Liang, J. Y., and Lipscomb, W. N. (1990) Binding of substrate CO2 to the active site of human carbonic anhydrase II: a molecular dynamics study, Proceedings of the National Academy of Sciences of the United States of America 87 3675 3679. 54. Domsic, J. F., Avvar u, B. S., Kim, C. U., Gruner, S. M., Agbandje McKenna, M., Silverman, D. N., and McKenna, R. (2008) Entrapment of carbon dioxide in the active site of carbonic anhydrase II, The Journal of biological chemistry 283 30766 30771.

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159 55. Sjoblom, B., Polentarutt i, M., and Djinovic Carugo, K. (2009) Structural study of X ray induced activation of carbonic anhydrase, Proceedings of the National Academy of Sciences of the United States of America 106 10609 10613. 56. Kim, C. U., Kapfer, R., and Gruner, S. M. (2005 ) High pressure cooling of protein crystals without cryoprotectants, Acta crystallographica. Section D, Biological crystallography 61 881 890. 57. Xue, Y., Vidgren, J., Svensson, L. A., Liljas, A., Jonsson, B. H., and Lindskog, S. (1993) Crystallographic analysis of Thr 200 ->His human carbonic anhydrase II and its complex with the substrate, HCO3, Proteins 15 80 87. 58. Huang, S., Sjblom, B., Sauer Eriksson, A. E., and Jonsson, B. H. (2002) Organization of an Efficient Carbonic Anhydrase: Implication s for the Mechanism Biochemistry 41 7628 7635. 59. West, D., Kim, C. U., Tu, C., Robbins, A. H., Gruner, S. M., Silverman, D. N., and McKenna, R. (2012) Structural and Kinetic Effects on Cha nges in the CO(2) Binding Pocket of Human Carbonic Anhydrase II, Biochemistry 60. Fisher, S. Z., Kovalevsky, A. Y., Domsic, J. F., Mustyakimov, M., McKenna, R., Silverman, D. N., and Langan, P. A. (2010) Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer, Biochemistry 49 415 421. 61. Fisher, S. Z., Aggarwal, M., Kovalesky, A., Silverman, D. N., and McKenna, R. (2012) Neutron diffraction of acetazolamide bound human carbonic anhydrase II reveals atomic details of drug bind ing, Journal of the American Chemical Society 134 14726 14729. 62. Roy, A., and Taraphder, S. (2007) Identification of proton transfer pathways in human carbonic anhydrase II, Journal of Physical Chemistry B 111 10563 10576. 63. Maupin, C. M., and Voth G. A. (2010) Proton transport in carbonic anhydrase: Insights from molecular simulation, Biochimica Et Biophysica Acta Proteins and Proteomics 1804 332 341. 64. Maupin, C. M., McKenna, R., Silverman, D. N., and Voth, G. A. (2009) Elucidation of the Pro ton Transport Mechanism in Human Carbonic Anhydrase II, Journal of the American Chemical Society 131 7598 7608. 65. Cui, Q., and Karplus, M. (2003) Is a "proton wire" concerted or stepwise? A model study of proton transfer in carbonic anhydrase, Journal of Physical Chemistry B 107 1071 1078.

PAGE 160

160 66. Fisher, S. Z., Tu, C. K., Bhatt, D., Govindasamy, L., Agbandje McKenna, M., McKenna, R., and Silverman, D. N. (2007) Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effec t of hydrophobic amino acid substitution in the active site of human carbonic anhydrase II, Biochemistry 42 3803 3813. 67. Maupin, C. M., Saunders, M. G., Thorpe, I. F., McKenna, R., Silverman, D. N., and Voth, G. A. (2008) Origins of enhanced proton tra nsport in the Y7F mutant of human carbonic anhydrase II, Journal of the American Chemical Society 130 11399 11408. 68. Avvaru, B. S., Kim, C. U., Sippel, K. H., Gruner, S. M., Agbandje McKenna, M., Silverman, D. N., and McKenna, R. (2010) A short, strong hydrogen bond in the active site of human carbonic anhydrase II, Biochemistry 49 249 251. 69. Bao, L., and Trachtenberg, M. C. (2006) Facilitated transport of CO2 across a liquid membrane: Comparing enzyme, amine, and alkaline, J Mem Sci 280 330 334. 70. Trachtenberg, M. C., Tu, C., Landers, R. A., Willson, R. C., McGregor, M. L., Laipis, P. J., Kennedy, J. F., Paterson, M., Silverman, D., Thomas, D., Smith, R. L., and Rudolph, F. B. (1999) Carbon Dioxide Transport By Proteic And Facilitated Transport Membranes, Life Support Biosph Sci 6 293 302. 71. Cowan, R. M., Ge, J., Qin, Y. J., McGregor, M. L., and Trachtenberg, M. C. (2003) CO2 capture by means of an enzyme based reactor, Ann N.Y. Acad Sci 984 453 469. 72. Esteban, A., Anzueto, A., Frutos, F. Alia, I., Brochard, L., Stewart, T. E., Benito, S., Epstein, S. K., Apezteguia, C., Nightingale, P., Arroliga, A. C., and Tobin, M. J. (2002) Characteristics and outcomes in adult patients receiving mechanical ventilation: a 28 day international study, J AMA 287 345 355. 73. Haft, J. W., Griffith, B. P., Hirschl, R. B., and Bartlett, R. H. (2002) Results of an artificial lung survey to lung transplant program directors, J Heart Lung Transplant. 21 467 473. 74. Maggiore, S. M., Richard, J. C., and Broch ard, L. (2003) What has been learnt from P/V curves in patients with acute lung injury/acute respiratory distress syndrome, European Respiratory Journal 22 22s 26s. 75. Ware, L. B., and Matthay, M. A. (2000) The acute respiratory distress syndrome, The N ew England journal of medicine 342 1334 1349.

PAGE 161

161 76. Arazawa, D. T., Oh, H. I., Ye, S. H., Johnson Jr, C. A., Woolley, J. R., Wagner, W. R., and Federspiel, W. J. (2012) Immobilized carbonic anhydrase on hollow fiber membranes accelerates CO 2 removal from blood, J. Membr. Sci. 403 404 25 31. 77. Kimmel, J. D., Arazawa, D. T., Ye, S. H., Shankarraman, V., Wagner, W. R., and Federspiel, W. J. (2013) Carbonic anhydrase immobilized on hollow fiber membranes using glutaraldehyde activated chitosan for artifici al lung applications, Journal of materials science. Materials in medicine 78. Beckley, P. D., Holt, D. W., and Tallman, R. D. (1995) Oxygenators for Extracorporeal Circulation, In Cardiopulmonary Bypass: Principles and Techniques of Extracorporeal Circul ation (Mora, C. T., Ed.), pp 199 219, Springer Verlag, New York. 79. Federspiel, W. J., and Henchir, K. A. (2004) Lung, Artificial: Basic Principles and Current Applications, In Encyclopedia of biomaterials and biomedical engineering (Wnek, G. E., and Bow lin, G. L., Eds.), pp 910 921, Marcel Dekker, New York. 80. Hattler, B. G., and Federspiel, W. J. (2002) Gas exchange in the venous system: Support for the failing lung, In The Artificial Lung (Vaslef, S. N., and Anderson, R. W., Eds.), pp 133 174, Landes Bioscience, Georgetown. 81. Okamoto, T., Tashiro, M., Sakanashi, Y., Tanimoto, H., Imaizumi, T., Sugita, M., and Terasaki, H. (1998) A New Heparin Bonded Dense Membrane Lung Combined with Minimal Systemic Heparinization Prolonged Extracorporeal Lung Assi st in Goats, Artificial Organs 22 864 872. 82. Watnabe, H., Hayashi, J., Ohzeki, H., Moro, H., Sugawara, M., and Eguchi, S. (1999) Biocompatibility of a silicone coated polypropylene hollow fiber oxygenator in an in vitro model, Ann Thorac Surg. 67 1315 1319. 83. Wegner, J. A. (1997) Oxygenator anatomy and function, Journal of cardiothoracic and vascular anesthesia 11 275 281. 84. Kaar, J. L., Oh, H. I., Russell, A. J., and Federspiel, W. J. (2007) Towards Improved Artificial Lungs Through Biocatalysi s, Biomaterials 28 3131 3139. 85. Bian, Y., Rong, Z., and Chang, T. M. (2012) Polyhemoglobin superoxide dismutase catalase carbonic anhydrase: a novel biotechnology based blood substitute that transports both oxygen and carbon dioxide and also acts as an antioxidant, Artif Cells Blood Substit Immobil Biotechnol 40 28 37.

PAGE 162

162 86. Gould, S. A., Moore, E. E., Hoyt, D. B., Ness, P. M., Norris, E. J., Carson, J. L., Hides, G. A., Freeman, I. H., DeWoskin, R., and Moss, G. S. (2002) The life sustaining capacity of human polymerized hemoglobin when red cells might be unavailable, J Am Coll Surg 195 445 452. 87. Satav, S. S., Bhat, S., and Thayumanavan, S. (2010) Feedback regulated drug delivery vehicles: carbon dioxide responsive cationic hydrogels for antidote release, Biomacromol 11 1735 1740. 88. Roskos, K. V., Fritzinger, B. K., Tefft, J. A., Nakayama, G. R., and Heller, J. (1995) Biocompatibility and in vivo morphine diffusion into a placebo morphine triggered naltrexone delivery device in rabbits, Biomat erials 16 1235 1239. 89. Bond, G. M., Medina, M. G., Stringer, J., and Simsek, E. F. A. (1999) Enzymatic Catalysis and CO2 Sequestration, World Res Rev 11 603 619. 90. Simsek Ege, F. A., Bond, G. M., and Stringer, J. (2002) Matrix molecular weight cut off for encapsulation of carbonic anhydrase in polyelectrolyte beads, Journal of Biomaterials Science, Polymer Edition 13 1175 1187. 91. Hassan, C. M., Doyle, F. J., and Peppas, N. A. (1997) Dynamic Behavior of Glucose Responsive Poly(methacrylic acid g ethylene glycol) Hydrogels, Macromolecules 30 6166 6173. 92. Han, D., Boissiere, O., Kumar, S., Tong, X., Tremblay, L. N., and Zhao, Y. (2012) Two way CO2 switchable triblock copolymer hydrogels, Macromol 45 7440 7445. 93. Fisher, Z., Boone, C. D., Bis was, S. M., Venkatakrishnan, B., Aggarwal, M., Tu, C., Agbandje McKenna, M., Silverman, D., and McKenna, R. (2012) Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II, Protein engineering, design & selection : PEDS 25 347 355. 94. Fulke, A. B., Mudliar, S. N., Yadav, R., Shekh, A., Srinivasan, N., Ramanan, R., and Chakrabarti, T. (2010) Bio mitigation of CO 2 calcite formation and simultaneous biodiesel precursors production using Chlorella sp., Bioresour. Te chnol. 101 8473 8476. 95. Ramanan, R., Kannan, K., Deshkar, A., Yadav, R., and Chakrabarti, T. (2010) Enhanced algal CO 2 sequestration through calcite deposition by Chlorella sp. and Spirulina platensis in a mini raceway pond, Bioresour. Technol. 101 26 16 2622.

PAGE 163

163 96. Hunt, J. A., Lesburg, C. A., Christianson, D. W., Thompson, R. B., and Fierke, C. A. (2000) Active site engineering of carbonic anhydrase and its application to biosensors., In The Carbonic Anhydrases: New Horizons (Chegwidden, W. R., Carte r, N. D., and Edwards, Y. H., Eds.), pp 221 240, Birkhuser Verlag, Boston, USA. 97. Kanbar, B., and Ozdemir, E. (2010) Thermal stability of carbonic anhydrase immobilized within polyurethane foam, Biotechnol. Prog. 26 1474 1480. 98. Hansen, J., Sato, M ., Ruedy, R., Lacis, A., and Oinas, V. (2000) Global warming in the twenty first century: an alternative scenario, Proceedings of the National Academy of Sciences of the United States of America 97 9875 9880. 99. EPA. (2007) Recent Climate Change: Atmosp heric Changes, In Climate Change Science Program United States Environmental Protection Agency, http://www.epa.gov/climatechange/science/indicators/index.html. 100. Shukman, D. (2013) Carbon dioxide passes symbolic mark, British Broadcasting Corporation, http://www.bbc.co.uk/news/science environment 22486153. 101. Luthi, D., Le Floch, M., Bereiter, B., Blunier, T., Barnola, J. M., Siegenthaler, U., Raynaud, D., Jouzel, J., Fischer, H., Kawamura, K., and Stocker, T. F. (2008) High resolution carbon dioxid e concentration record 650,000 800,000 years before present, Nature 453 379 382. 102. Petit, J. R., Jouzel, J., Raynaud, D., Barkov, N. I., Barnola, J. M., Basile, I., Bender, M., Chappellaz, J., Davis, M., Delaygue, G., Delmotte, M., Kotlyakov, V. M., L egrand, M., Lipenkov, V. Y., Lorius, C., Ppin, L., Ritz, C., Saltzman, E., and Stievenard, M. (1999) Climate and atmospheric history of the past 420,000 years from the Vostok ice core, Antarctica, Nature 399 429 436. 103. Siegenthaler, U., Stocker, T. F ., Monnin, E., Luthi, D., Schwander, J., Stauffer, B., Raynaud, D., Barnola, J. M., Fischer, H., Masson Delmotte, V., and Jouzel, J. (2005) Stable carbon cycle climate relationship during the Late Pleistocene, Science 310 1313 1317. 104. Spahni, R., Chap pellaz, J., Stocker, T. F., Loulergue, L., Hausammann, G., Kawamura, K., Fluckiger, J., Schwander, J., Raynaud, D., Masson Delmotte, V., and Jouzel, J. (2005) Atmospheric methane and nitrous oxide of the Late Pleistocene from Antarctic ice cores, Science 3 10 1317 1321. 105. Pearson, P. N., and Palmer, M. R. (2000) Atmospheric carbon dioxide concentrations over the past 60 milliong years, Nature 406 695 699.

PAGE 164

164 106. Houghton, J. T., Ding, Y., Griggs, D. J., Noguer, M., van der Linden, P. J., Dai, X., and M askell, K. (2001) IPCC, 2001: Climate Change 2001: The Scientific Basis. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change, p 881, Cambridge University Press, Cambridge, United Kingdom and New Y ork, NY, USA. 107. Weart, S. (2011) The Carbon Dioxide Greenhouse Effect, In The Discovery of Global Warming American Institue of Physics, http://www.aip.org/history/climate/co2.htm. 108. Jansen, K. (2007) What Do Reconstructions Based on Palaeoclimatic Proxies Show?, pp 466 478. 109. IPCC. (2007) Synthesis Report Summary for Policymakers; Observed changes in climate and their effects. 110. Kennedy, C. (2012) State of the Climate: 2011 Global Sea Level, In ClimateWatch Magazine NOAA Climate Services P ortal. 111. Morello, L. (2010) Oceans Turn More Acidic Than Last 800,000 Years, In Scientific American http://www.scientificamerican.com/article.cfm?id=acidic oceans. 112. Benson, S. M., and Surles, T. (2006) Carbon Dioxide Capture and Storage: An Overv iew With Emphasis on Capture and Storage in Deep Geological Formations, Proceedings of the IEEE 94 1795 1805. 113. Pierre, A. C. (2012) Enzymatic Carbon Dioxide Capture, In ISRN Chemical Engineering pp 1 22. 114. Boone, C. D., Gill, S., Habibzadegan, A ., and McKenna, R. (2013) Carbonic anhydrases and their industrial applications, Current Topics in Biochemical Research 14 1 10. 115. Zevenhoven, R., Eloneva, S., and Teir, S. (2006) Chemical fixation of CO2 in carbonates: Routes to valuable products and long term storage, Catalysis Today 115 73 79. 116. Allen, D. J., and Brent, G. F. (2010) Sequestering CO(2) by mineral carbonation: stability against acid rain exposure, Environ Sci Technol 44 2735 2739. 117. Astachov, L., Nevo, Z., Brosh, T., and Vag o, R. (2011) The structural, compositional and mechanical features of the calcite shell of the barnacle Tetraclita rufotincta, Journal of structural biology 175 311 318.

PAGE 165

165 118. Suzuki, M., Saruwatari, K., Kogure, T., Yamamoto, Y., Nishimura, T., Kato, T., a nd Nagasawa, H. (2009) An acidic matrix protein, Pif, is a key macromolecule for nacre formation, Science 325 1388 1390. 119. Arakawa, H., Aresta, M., Armor, J. N., Barteau, M. A., Beckman, E. J., Bell, A. T., Bercaw, J. E., Creutz, C., Dinjus, E., Dixon D. A., Domen, K., DuBois, D. L., Eckert, J., Fujita, E., Gibson, D. H., Goddard, W. A., Goodman, D. W., Keller, J., Kubas, G. J., Kung, H. H., Lyons, J. E., Manzer, L. E., Marks, T. J., Morokuma, K., Nicholas, K. M., Periana, R., Que, L., Rostrup Nielson J., Sachtler, W. M. H., Schmidt, L. D., Sen, A., Somorjai, G. A., Stair, P. C., Stults, B. R., and Tumas, W. (2001) Catalysis Research of Relevance to Carbon Management: Progress, Challenges, and Opportunities, Chemical Reviews 101 953 996. 120. Beckm an, E. J. (1999) POLYMER SYNTHESIS:Enhanced: Making Polymers from Carbon Dioxide, Science 283 946 947. 121. Sakakura, T., Choi, J. C., and Yasuda, H. (2007) Transformation of carbon dioxide, Chem Rev 107 2365 2387. 122. Chen, P., Min, M., Chen, Y., Wan g, L., Li, Y., Wang, C., Wan, Y., Wang, X., Cheng, Y., Deng, S., Hennessy, K., Lin, X., Liu, Y., Wang, Y., Martinez, B., and Ruan, R. (2009) Review of the biological and engineering aspects of algae to fuels approach, Int J Argic & Biol Eng 2 1 30. 123. Briggs, M. (2004) Widescale Production from Algae, The University of New Hampshire, Physics Department. 124. (April 29, 2013) Monthly Biodiesel Production Report, U.S. Energy Information Administration, www.eia.gov. 125. V., M. B., Samiei, H., and Cheng, K. (2007) Biofuel demand pushes up food prices, In IMF Survey Magazine IMF Research Department. 126. Boddiger, D. (2007) Boosting biofuel crops could threaten food security, The Lancet 370 923 924. 127. Jeong, M. J., Gillis, J. M., and Hwang, J. Y. (2 003) Carbon dioxide mitigation by microalgal photosynthesis, Bull. Korean Chem. Soc. 24 1763 1766. 128. Johnson, M. B., and Wen, Z. (2009) Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal bio mass, Energy Fuels 23 5179 5183.

PAGE 166

166 129. Lopez, C. V. G., Fernandez, F. G. A., Sevilla, J. M. F., Fernandez, J. F. S., Garcia, M. C. C., and Grima, E. M. (2009) Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in carbon dioxide removal processes, Bioresour. Technol. 100 5904 5910. 130. Gonzalez Fernandez, C., and Ballesteros, M. (2012) Linking microalgae and cyanobacteria culture conditions and key enzymes for carbohydrate accumulation, Biotechnology advances 30 1655 1661. 131. Pires, J. C. M., Alvim Ferraz, M. C. M., Martins, F. G., and Simes, M. (2012) Carbon dioxide capture from flue gases using microalgae: Engineering aspects and biorefinery concept, Renewable and Sustainable Energy Reviews 16 3043 3053. 132. Badger, M. R. (2003) CO2 conc entrating mechanisms in cyanobacteria: molecular components, their diversity and evolution, Journal of Experimental Botany 54 609 622. 133. Bloch, M. R., Sasson, J., Ginzburg, M. E., Goldman, Z., Ginzburg, B. Z., Garti, N., and Porath, A. (1982) Oil Prod uction From Algae, USA. 134. Ellis, R. J. (2010) Biochemistry: Tackling unintelligent design, Nature 463 164 165. 135. Shekh, A. Y., Krishnamurthi, K., Mudliar, S. N., Yadav, R. R., Fulke, A. B., Devi, S. S., and Chakrabarti, T. (2012) Recent Advancemen ts in Carbonic Anhydrase Driven Processes for CO2Sequestration: Minireview, Critical Reviews in Environmental Science and Technology 42 1419 1440. 136. Aggarwal, M., Boone, C. D., Kondeti, B., Tu, C., Silverman, D. N., and McKenna, R. (2013) Effects of c ryoprotectants on the structure and thermostability of the human carbonic anhydrase II acetazolamide complex, Acta crystallographica. Section D, Biological crystallography 69 860 865. 137. Sippel, K. H., Robbins, A. H., Domsic, J., Genis, C., Agbandje Mc Kenna, M., and McKenna, R. (2009) High resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design, Acta crystallographica. Section F, Structural biology and crystallization communications 65 992 995. 138. Rost, B., Richter, K. U., Riebesell, U. L. F., and Hansen, P. J. (2006) Inorganic carbon acquisition in red tide dinoflagellates, Plant, Cell and Environment 29 810 822.

PAGE 167

167 139. Bond, G. M., Medina, M. G., Stringer, J., and Simsek, E. F. A (2008) CO 2 Capture from Coal Fired Utility Generation Plant Exhausts, and Seqeustration by a Biomimetic Route Based on Enzymatic Catalysis, DOE, http://www.netl.doe.gov/publications/proceedings/01/carbon_seq/5a5.pdf. 140. da Costa Ores, J., Sala, L., Ce rveira, G. P., and Kalil, S. J. (2012) Purification of carbonic anhydrase from bovine erythrocytes and its application in the enzymic capture of carbon dioxide, Chemosphere 88 255 259. 141. Forsman, C., Behravan, G., Osterman, A., and Jonsson, B. H. (198 8) Production of active human carbonic anhydrase II in E. coli, Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry 42 314 318. 142. Bond, G. M., Stringer, J., Brandvold, D. K., Simsek, F. A., Medina, M. G., and Egeland, G. (2001) Dev elopment of Integrated System for Biomimetic CO2Sequestration Using the Enzyme Carbonic Anhydrase, Energy & Fuels 15 309 316. 143. Avvaru, B. S., Busby, S. A., Chalmers, M. J., Griffin, P. R., Venkatakrishnan, B., Agbandje McKenna, M., Silverman, D. N., and McKenna, R. (2009) Apo Human Carbonic Anhdrase II Revisited: Implications of the Loss of a Metal in Protein Structure, Stability, and Solvent Network, Biochemistry 48 7365 7372. 144. Jochens, H., Aerts, D., and Bornscheuer, U. T. (2010) Thermostabili zation of an esterase by alignment guided focussed directed evolution, Protein engineering, design & selection : PEDS 23 903 909. 145. Mrabet, N. T., Van den Broeck, A., Van den Brande, I., Stanssens, P., Laroche, Y., Lambeir, A. M., Matthijssens, G., Je nkins, J., and Chiadmi, M. (1992) Arginine residues as stabilizing elements in proteins, Biochemistry 31 2239 2253. 146. Strickler, S. S., Gribenko, A. V., Keiffer, T. R., Tomlinson, J., Reihle, T., Loladze, V. V., and Makhatadze, G. I. (2006) Protein st ability and surface electrostatics: a charged relationship, Biochemistry 45 2761 2766. 147. Filikov, A. V., Hayes, R. J., Luo, P., Stark, D. M., Chan, C., Kundu, A., and Dahiyat, B. I. (2002) Computational stabilization of human growth hormone, Protein s cience : a publication of the Protein Society 11 1452 1461. 148. Permyakov, S. E., Makhatadze, G. I., Owenius, R., Uversky, V. N., Brooks, C. L., Permyakov, E. A., and Berliner, L. J. (2005) How to improve nature: study of the electrostatic properties of the surface of alpha lactalbumin, Protein engineering, design & selection : PEDS 18 425 433.

PAGE 168

168 149. Potapov, V., Cohen, M., and Schreiber, G. (2009) Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details, Protein engineering, design & selection : PEDS 22 553 560. 150. Reetz, M. T., Carballeira, J. D., and Vogel, A. (2006) Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for Increasing Protein Thermostability, Angewand te Chemie 118 7909 7915. 151. Dill, K. A. (1990) Dominant forces in protein folding, Biochemistry 29 7133 7155. 152. Jaenicke, R. (1991) Protein stability and molecular adaptation to extreme conditions, European journal of biochemistry / FEBS 202 715 728. 153. Davies, G. J., Gamblin, S. J., Littlechild, J. A., and Watson, H. C. (1993) The structure of a thermally stable 3 phosphoglycerate kinase and a comparison with its mesophilic equivalent, Proteins 15 283 289. 154. Vieille, C., Hess, J. M., Kell y, R. M., and Zeikus, J. G. (1995) xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana, Applied and environmental microbiology 61 1867 1875. 155. Auerbach, G., Ostendorp, R., Prade, L., Korndrfer, I., Dams, T., Huber, R., and Jaenicke, R. (1998) Lactate dehydrogenase from the hyperthermophilic bacterium thermotoga maritima: the crystal structure at 2.1 A resolution reveals strategies for intrinsic protein stabilization, Structure 6 769 781. 156. Chi, Y. I., Martinez Cruz, L. A., Jancarik, J., Swanson, R. V., Robertson, D. E., and Kim, S. H. (1999) Crystal structure of the beta glycosidase from the hyperthermophile Thermosphaera aggregans: insights into its activity and thermostability, FEBS Lett. 445 375 383. 157. Hopfner, K. P., Eichinger, A., Engh, R. A., Laue, F., Ankenbauer, W., Huber, R., and Angerer, B. (1999) Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius, Proceedings of the National Academy of Scie nces of the United States of America 96 3600 3605. 158. Isupov, M. N., Fleming, T. M., Dalby, A. R., Crowhurst, G. S., Bourne, P. C., and Littlechild, J. A. (1999) Crystal structure of the glyceraldehyde 3 phosphate dehydrogenase from the hyperthermophil ic archaeon Sulfolobus solfataricus, J Mol Biol 291 651 660.

PAGE 169

169 159. Maes, D., Zeelen, J. P., Thanki, N., Beaucamp, N., Alvarez, M., Thi, M. H., Backmann, J., Martial, J. A., Wyns, L., Jaenicke, R., and Wierenga, R. K. (1999) The crystal structure of tri osephosphate isomerase (TIM) from Thermotoga maritima: a comparitive thermostability structural analysis of ten different TIM structures, Proteins 37 441 453. 160. Russell, R. J., Ferguson, J. M., Hough, D. W., Danson, M. J., and Taylor, G. L. (1997) The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolutoin, Biochemistry 36 9983 9994. 161. Tahirov, T. H., Oki, H., Tsukihara, T., Ogasahara, K., Yutani, K., Ogata, K., Izu, Y., Tsunasawa, S., and Kato, I. (1998) Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus, J Mol Biol 284 101 124. 162. Bauer, M. W., and Kelly, R. M. (1998) The family 1 beta glucosidases from Pyrococcus furiosus and Agrobacterium faecal is share a common catalytic mechanism, Biochemistry 37 17170 17178. 163. Zwickl, P., Fabry, S., Bogedain, C., Haas, A., and Hensel, R. (1990) Glyceraldehyde 3 phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: character ization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli, Journal of bacteriology 172 4329 4338. 164. Privalov, P. L., and Khechinashvili, N. N. (1974) A thermodynamic approach to the problem of stabilization of globu lar protein structure: a calorimetric study, J Mol Biol 86 665 684. 165. Kawamura, S., Kakuta, Y., Tanaka, I., Hikichi, K., Kuhara, S., Yamasaki, N., and Kimura, M. (1996) Glycine 15 in the bend between two alpha helices can explain the thermostability o f DNA binding protein HU from Bacillus stearothermophilus, Biochemistry 35 1195 1200. 166. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons, Prote ins 11 281 296. 167. Vieille, C., and Zeikus, G. J. (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability, Microbiology and molecular biology reviews : MMBR 65 1 43. 168. Matsumura, M., Signor, G., and Matthews, B. W. (1989) Substantial increase of protein stability by multiple disulphide bonds, Nature 342 291 293.

PAGE 170

170 169. Whitney, P. L., and Briggle, T. V. (1982) Membrane associated carbonic anhydrase purified from bovine lung, The Journal of biological chemistry 2 57 12056 12059. 170. Waheed, A., Okuyama, T., Heyduk, T., and Sly, W. S. (1996) Carbonic anhydrase IV: purification of a secretory form of the recombinant human enzyme and identification of the positions and importance of its disulfide bonds, Archives of biochemistry and biophysics 333 432 438. 171. Stams, T., Nair, S. K., Okuyama, T., Waheed, A., Sly, W. S., and Christianson, D. W. (1996) Crystal structure of the secretory form of membrane associated human carbonic anhydrase IV at 2.8 A resolution, Pro c. Natl. Acad. Sci. U.S.A. 93 13589 13594. 172. Mrtensson, L. G., Karlsson, M., and Carlsson, U. (2002) Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond, Biochemistry 41 15867 15875. 173. Samiot akis, A., Homouz, D., and Cheung, M. S. (2010) Multiscale investigation of chemical interference in proteins, The Journal of chemical physics 132 175101. 174. Wang, Q., Christiansen, A., Samiotakis, A., Wittung Stafshede, P., and Cheung, M. S. (2011) Com parison of chemical and thermal protein denaturation by combination of computational and experimental approaches. II, The Journal of chemical physics 135 175102. 175. Burley, S. K., and Petsko, G. A. (1985) Aromatic aromatic interaction: a mechanism of p rotein structure stabilization, Science 229 23 28. 176. Dong, G., Vieille, C., Savchenko, A., and Zeikus, J. G. (1997) Cloning, sequencing, and expression of the gene encoding extracellular alpha amylase from Pyrococcus furiosus and biochemical character ization of the recombinant enzyme, Applied and environmental microbiology 63 3569 3576. 177. Teplyakov, A. V., Kuranova, I. P., Harutyunyan, E. H., Vainshtein, B. K., Frmmel, C., Hhne, W. E., and Wilson, K. S. (1990) Crystal structure of thermitase at 1.4 A resolution, J. Mol. Biol. 214 261 279. 178. Ishikawa, K., Okumura, M., Katayanagi, K., Kimura, S., Kanaya, S., Nakamura, H., and Morikawa, K. (1993) Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution, J Mo l Biol 230 529 542. 179. Serrano, L., Bycroft, M., and Fersht, A. R. (1991) Aromatic aromatic interactions and protein stability. Investigation by double mutant cycles, J Mol Biol 218 465 475.

PAGE 171

171 180. Matthews, B. W., Nicholson, H., and Becktel, W. J. (198 7) Enhanced protein thermostability from site directed mutations that decrease the entropy of unfolding, Proc. Natl. Acad. Sci. U.S.A. 84 6663 6667. 181. Sriprapundh, D., Vieille, C., and Zeikus, J. G. (2000) Molecular determinants of xylose isomerase th ermal stability and activity: analysis of thermozymes by site directed mutagenesis, Protein engineering 13 259 265. 182. Nakai, T., Okada, K., Akutsu, S., Miyahara, I., Kawaguchi, S., Kato, R., Kuramitsu, S., and Hirotsu, K. (1999) Structure of Thermus t hermophilus HB8 aspartate aminotransferase and its complex with maleate, Biochemistry 38 2413 2424. 183. Li, C., Heatwole, J., Soelaiman, S., and Shoham, M. (1999) Crystal structure of a thermophilic alcohol dehydrogenase substrate complex suggests deter minants of substrate specficity and thermostability, Proteins 37 619 627. 184. Watanabe, K., Masuda, T., Ohashi, H., Mihara, H., and Suzuki, Y. (1994) Multiple proline substitutions cumulatively thermostabilze Bacillus cereus ATCC7064 oligo 1,6 glucosida se. Irrefragable proof supporting the proline rule, Eur. J. Biochem. 226 277 283. 185. Rose, G. D., Gierasch, L. M., and Smith, J. A. (1985) Turns in peptides and proteins, Advances in protein chemistry 37 1 109. 186. Manco, G., Giosu, E., D'Auria, S. Herman, P., Carrea, G., and Rossi, M. (2000) Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone sensitive subfamily from the archaeon Archaeoglobus fulgidus, Arch. Biochem. Biophys. 373 182 192. 187. Bonisch, H., Backmann, J., Kath, T., Naumann, D., and Schafer, G. (1996) Adenylate kinase from Sulfolobus acidocaldarius: expression in Escherichia coli and characterization by Fourier transform infrared spectroscopy, Archives of bioch emistry and biophysics 333 75 84. 188. Jaenicke, R., and Bohm, G. (1998) The stability of proteins in extreme environments, Current opinion in structural biology 8 738 748. 189. Zvodszky, P., Kardos, J., Svingor, and Petsko, G. A. (1998) Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins, Proc. Natl. Acad. Sci. U.S.A. 95 406 411. 190. Gershenson, A., Schauerte, J. A., Giver, L., and Arnold, F. H. (2000) Tryptophan phophorescence study of enzyme flexibility a nd unfolding in laboratory evolved thermostable esterases, Biochemistry 39 4658 4665.

PAGE 172

172 191. Villbrandt, B., Sagner, G., and Schomburg, D. (1997) Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments, Pro tein engineering 10 1281 1288. 192. Khalifah, R. G., Strader, D. J., Bryant, S. H., and Gibson, S. M. (1977) Carbon 13 nuclear magnetic resonance probe of active site ionizations in human carbonic anhydrase B, Biochemistry 16 2241 2247. 193. Otwinowski Z., and Minor, W. (1997) [20] Processing of X ray diffraction data collected in oscillation mode, Methods Enzymol. 276 307 326. 194. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G J., Grosse Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python based system for macromolecular structure solution, Act a crystallographica. Section D, Biological crystallography 66 213 221. 195. Emsley, P., and Cowtan, K. (2004) Coot: model building tools for molecular graphics, Acta crystallographica. Section D, Biological crystallography 60 2126 2132. 196. Becktel, W J., and Schellman, J. A. (1987) Protein stability curves, Biopolymers 26 1859 1877. 197. Silverman, D. N. (1982) Carbonic anhydrase: oxygen 18 exchange catalyzed by an enzyme with rate contributing proton transfer steps, Methods Enzymol 87 732 752. 1 98. Ippolito, J. A., Nair, S. K., Alexander, R. S., Kiefer, L. L., Fierke, C. A., and Christia new crystalline form reveals a partially occupied zinc binding site, "Protein Engineering, Design and Selection" 8 975 980. 199. Lloyd, M. D., Pederick, R. L., Natesh, R., Woo, L. W., P urohit, A., Reed, M. J., Acharya, K. R., and Potter, B. V. (2005) Crystal structure of human carbonic anhydrase II at 1.95 A resolution in complex with 667 coumate, a novel anti cancer agent, The Biochemical journal 385 715 720. 200. Stams, T., and Chris tianson, D. W. (2000) X ray crystallographic studies of mammalian carbonic anhdyrase isozymes, In The Carbonic Anhydrases: New Horizons (Chegwidden, W. R. C., N.D. ; Edwards, Y.H., Ed.), pp 159 174, Birkhauser Verlag, Basel, Switzerland.

PAGE 173

173 201. Huang, S., Xue, Y., Sauer Eriksson, E., Chirica, L. C., Lindskog, S., and Jonsson, B. H. (1998) Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazoliamide, J Mol Biol 283 301 310. 202. Elleby, B., Chirica, L C., Tu, C., Zeppezauer, M., and Lindskog, S. (2001) Characterization of carbonic anhydrase from Neisseria gonorrhoeae, European Journal of Biochemistry 268 1613 1619. 203. Di Fiore, A., Capasso, C., De Luca, V., Monti, S. M., Carginale, V., Supuran, C. T., Scozzafava, A., Pedone, C., Rossi, M., and De Simone, G. (2013) X ray structure of the first `extremo alpha carbonic anhydrase', a dimeric enzyme from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1, Acta crystallographica. Sect ion D, Biological crystallography 69 1150 1159. 204. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures, Journal of Applied Crystallography 26 283 291. 205. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA. 206. Boone, C. D., Habibzadegan, A., Tu, C., Silverman, D. N., and McKenna, R. (2013) Structural and catalytic characterization of a thermally stable and aci d stable variant of human carbonic anhydrase II containing an engineered disulfide bond, Acta crystallographica. Section D, Biological crystallography 69 1414 1422. 207. Schmidt, B., Ho, L., and Hogg, P. J. (2006) Allosteric disulfide bonds, Biochemistry 45 7429 7433. 208. Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C., Alagona, G., Profeta, S. J., and Weinger, P. (1984) A new force field for molecular simulation of nucleic acis and protein, Journal of the American Chemical Society 106 765 784. 209. Katz, B. A., and Kossiakoff, A. (1986) The crystallographically determined structures of atypical strained disulfides engineered into subtilisin, The Journal of biological chemistry 261 15480 15485. 210. Wells, J. A., and Powers, D. B. (19 86) In vivo formation and stability of engineered disulfide bonds in subtilisin, The Journal of biological chemistry 261 6564 6570.

PAGE 174

174 211. Kuwajima, K., Ikeguchi, M., Sugawara, M., Hiraoka, Y., and Sugai, S. (1990) Kinetics of disulfide bond reduction in a lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6 Cys120 disulfide bond, Biochemistry 29 8240 8249. 212. Wetzel, R., Perry, L. J., Baase, W. A., and Becktel, W. J. (1988) Disulfide bonds and thermal stability in T4 lysozyme, Proceedings of the National Academy of Sciences of the United States of America 85 401 405. 213. Pjura, P. E., Matsumura, M., Wozniak, J. A., and Matthews, B. W. (1990) Structure of a thermostabile disulfide bridge mutant of phage T4 lysozyme shows that an engineered cross link in a flexible region does not increase the rigidity of the folded protein, Biochemistry 29 2592 2598. 214. Cleland, J. L., and Wang, I. C. (1990) Refolding and aggregation of bovine carbonic anhdyrase B: Quasi elastic light scatte ring analysis, Biochemistry 29 11072 11078. 215. Dombkowski, A. A. (2003) Disulfide by Design: a computational method for the rational design of disulfide bonds in proteins, Bioinformatics 19 1852 1853. 216. Chen, V. B., Arendall, W. B., 3rd, Headd, J. J ., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all atom structure validation for macromolecular crystallography, Acta crystallographica. Section D, Biological crystallography 66 12 21. 217. Boone, C. D., Gill, S., Tu, C., Silverman, D. N., and McKenna, R. (2013) Structural, catalytic and stabilizing consequences of aromatic cluster variants in human carbonic anhydrase II, Archives of biochemistry and biophysics 539 31 37. 218. N ovoa, J. J., and Mota, F. (2000) The C hydrogen bonded nature: a theoretical study, Chemical Physics Letters 318 345 354. 219. Hansson, M. D., Rzeznicka, K., Rosenback, M., Hansson, M., and Sirijovski, N. (2008) PCR mediated deletion of plasmid DNA, Analytical biochemistry 375 373 375. 220. Milov, D. E., Jou, W. S., Shireman, R. B., and Chun, P. W. (1992) The effect of bile salts on carbonic anhydrase, Hepatology 15 288 296. 221. Staels, B., and Fonseca, V. A. ( 2009) Bile acids and metabolic regulation: mechanisms and clinical responses to bile acid sequestration, Diabetes care 32 Suppl 2 S237 245.

PAGE 175

175 222. Salomoni, M., Zuccato, E., Granelli, P., Montorsi, W., Doldi, S. B., Germiniani, R., and Mussini, E. (1989) Ef fect of bile salts on carbonic anhydrase from rat and human gastric mucosa, Scand J Gastroenterol 24 28 32. 223. Kivilaakso, E. (1982) Inhibition of gastric mucosal carbonic anhydrase by taurocholic acid and other ulcerogenic agents, American journal of s urgery 144 554 557. 224. Benedetti, A., Di Sario, A., Marucci, L., Svegliati Baroni, G., Schteingart, C. D., Ton Nu, H. T., and Hofmann, A. F. (1997) Carrier mediated transport of conjugated bile acids across the basolateral membrane of biliary epithelial cells, The American journal of physiology 272 G1416 1424. 225. Choquet Kastylevsky, G., Vial, T., and Descotes, J. (2002) Allergic adverse reactions to sulfonamides, Current Allergy and Asthma Reports 2 16 25. 226. Adams, P. D., Afonine, P. V., Bunkoczi G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python based system for macromolecular structu re solution, Acta Cryst D66 213 221. 227. Emsley, P., and Cowtan, K. (2004) Coot: model building tools for molecular graphics, Acta Cryst D60 2126 2132. 228. Fisher, S. Z., Aggarwal, M., Kovalevsky, A. Y., Silverman, D. N., and McKenna, R. (2012) Neutron diffraction of acetazolamide bound human carbonic anhydrase II reveals atomic details of drug binding, Journal of the American Chemical Society 134 14726 14729. 229. Alterio, V., Hilvo, M., Di Fiore, A., Supuran, C. T., Pan, P., Parkkila, S., Scaloni, A. Pastorek, J., Pastorekova, S., Pedone, C., Scozzafava, A., Monti, S. M., and De Simone, G. (2009) Crystal structure of the catalytic domain of the tumor associated human carbonic anhydrase IX, Proceedings of the National Academy of Sciences of the United States of America 106 16233 16238. 230. Whittington, D. A., Waheed, A., Ulmasov, B., Shah, G. N., Grubb, J. H., Sly, W. S., and Christianson, D. W. (2001) Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic me mbrane protein overexpressed in certain cancer tumor cells, Proceedings of the National Academy of Sciences of the United States of America 98 9545 9550.

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176 BIOGRAPHICAL SKETCH Chris was born on a cool, summer morning in late July in the foothills of No rth Carolina. As a child, he enjoyed the comfort of the outdoors where he would take refuge in a freshly built clubhouse in the middle of the forest or floating down a river in an inner tube. The childhood dreams of becoming either a professional wrestler or astronaut were extinguished upon learning of the Krebs c ycle in high school. The idea of how one single molecule can be chemically converted into so many useful byproducts fascinated him so much that he decided to study biochemistry at the University o f North Carolina at Charlotte. After succe ssfully graduating with two Bachelor of Science degrees in biochemistry and cell p hysio logy in 2006, Chris joined the m aster s program in the Department of Chemistry where he studied the biophysical interactions of the HIV 1 envelope glycoprotein gp120 and the T cell receptor CD4, and later graduated in 2009. Chris joined the Department of Biochemistry & Molecular Biology in the College of Medicine at the University of Florida in 2010 and received his Doctor of Philo sophy in biomedical sciences in 2014 His hope after a post doctoral position is to start a research lab of his own where he can utilize all of the biophysical techniques he has learned to elucidate the structure and function of proteins.


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