<%BANNER%>

An Electron Paramagnetic Resonances Study of Surfactant Protein B Mimic Kl4

Permanent Link: http://ufdc.ufl.edu/UFE0044133/00001

Material Information

Title: An Electron Paramagnetic Resonances Study of Surfactant Protein B Mimic Kl4
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Turner, Austin L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: epr -- kl4 -- lung -- nmr -- pulmonary -- saturation -- surfactant
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: KL4 is a 21 amino acid peptide used to mimic the C-terminus of lung surfactant protein B, a protein known to lower the surface tension in the highly dynamic alveoli. Understanding how KL4 interacts with lipid vesicles of varying composition will provide insight into potential treatment for diseases such as respiratory distress syndrome. Recent 31P and 2H NMR studies have shown that KL4 binds differently to POPC:POPG and DPPC:POPG multilamellar vesicles, with the latter being found at elevated levels in lung surfactants. The current study uses electron paramagnetic resonance spectroscopy (EPR) and a technique called power saturation to study the effects of KL4 binding to lipid bilayers both at the lipid and peptide level. Power saturation can be used to determine a change in the accessibility of the spin label to molecular oxygen in the bilayer interior and to NiAA, an aqueous soluble nickel complex. Using information gathered from these experiments we will provide insights into the depth and orientation of the peptide within different bilayer systems.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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.
Statement of Responsibility: by Austin L Turner.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Fanucci, Gail E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044133:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044133/00001

Material Information

Title: An Electron Paramagnetic Resonances Study of Surfactant Protein B Mimic Kl4
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Turner, Austin L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: epr -- kl4 -- lung -- nmr -- pulmonary -- saturation -- surfactant
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: KL4 is a 21 amino acid peptide used to mimic the C-terminus of lung surfactant protein B, a protein known to lower the surface tension in the highly dynamic alveoli. Understanding how KL4 interacts with lipid vesicles of varying composition will provide insight into potential treatment for diseases such as respiratory distress syndrome. Recent 31P and 2H NMR studies have shown that KL4 binds differently to POPC:POPG and DPPC:POPG multilamellar vesicles, with the latter being found at elevated levels in lung surfactants. The current study uses electron paramagnetic resonance spectroscopy (EPR) and a technique called power saturation to study the effects of KL4 binding to lipid bilayers both at the lipid and peptide level. Power saturation can be used to determine a change in the accessibility of the spin label to molecular oxygen in the bilayer interior and to NiAA, an aqueous soluble nickel complex. Using information gathered from these experiments we will provide insights into the depth and orientation of the peptide within different bilayer systems.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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.
Statement of Responsibility: by Austin L Turner.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Fanucci, Gail E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044133:00001


This item has the following downloads:


Full Text

PAGE 1

1 AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF SURFACTANT PROTEIN B MIMIC KL 4 By AUSTIN LISLE TURNER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

PAGE 2

2 2012 Austin Lisle Turner

PAGE 3

3 To my parents Randall and Kathleen Turner and my brothers Sean and Ashton Turner w ith special thanks to my grandmother Virginia Hawley, my godparents Jim and Sue Lami e, and my friends along the way

PAGE 4

4 ACKNOWLEDGMENTS First and foremost, I would like to thank my parents Kathleen and Randall Turner and my brothers Sean and Ashton for their sacrifices, support, patience and love. Without them, none of this work would have been possible. Secondly, I want to express my deepest gratitude to my advisor and mentor, Doctor Gail E. Fanucci for her patience and encouragement, for countless opportunities to present my research at national conferences, and for her g uidance and support. I would also like to offer my sincere gratitude to Doctor Joanna Long for her collaboration and insight on the surfactant project, in addition to welcoming me into her group as one of her own. I would also like to thank all the other members of my doctoral committee, Doctors Alex Angerhofer, Nicole Horenstein, and Ben Smith. I would like to thank Ben Smith for his acceptance and guidance throughout this Ph.D. process, Alex Angerhofer for his EPR expertise along the way, Nicole Horens tein for her help at seeing the big picture of my Ph.D. project along with giving me the opportunity to teach alongside her in Biochemistry Lab. I would like to thank all past and present members of the Fanucci and Long groups for their friendship, help, and support, particularly Jeffrey Carter, Adam Smith, Suzanne Farver, Anna Kuznetsova and former members Doctors Jamie Kear, Luis Galiano, Jordan Mathias, Mandy Blackburn, and Natasha Pirman. Each of you played an important part in making graduate school more enjoyable and providing me with the memories that will last forever. In addition, thanks goes to former undergraduate Phil Goff who studied in the Fanucci group and helped keep me sane during the long EPR hours.

PAGE 5

5 I would like to thank the Department of Chemistry at Bradley University, where I obtained my passion and love for chemistry. In particular I want to express my deepest gratitude to Doctor Max Taylor who without knowing it turned an electrical engineering student into a lifelong chemist by sh owing him his love and passion for chemistry. I would also like to thank the Department of Chemistry at the University of Wisconsin at Whitewater, where I finished my pursuit to obtain my Bachelor of Science (B.S.) degree in chemistry. In particular, I w ant to express my sincere gratitude to Doctor Kathy Asala for her mentoring and guidance along the way. I would like to thank Doctor Ken Matuszak for his endless mentoring and countless scientific discussions while working at Abbott Laboratories and his co ntinued friendship Florida. In addition, I would like to thank the many friends and colleagues from Abbott Laboratories who went above and beyond to see to it that I would have the best possible opportunities in my scientific endeavors. A special thanks to Doctor Rick Yost who originally brought me into his analytical group and helped lead my transition into the Biochemistry division. I would like to thank the University of Florida Alumni Fellowship program and the University of Florida Startup for funding and the University of Physics department and Doctor Steve Hagan for use and his knowledge on his circular dichroism instrument. This work was supported by NIH R01 GM07 7232 and NIH 1R01HL076586.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 16 CHAPTER 1 INTRODUCTION TO PULMONARY SURFACTANTS ................................ ........... 17 Surfactants ................................ ................................ ................................ .............. 17 Properties of Surfactants ................................ ................................ .................. 18 General Mechanism of Surfactant Function ................................ ..................... 19 Introduction to Pulmonary Surfactants ................................ ................................ .... 20 Pulmonary Surfactant Deficiency Diseases ................................ ...................... 22 Respiratory Distress Syndrome ................................ ................................ ........ 23 Surfactant Lipids ................................ ................................ ............................... 25 Surfactant Proteins ................................ ................................ ........................... 26 Surfactant Protein B and Analogs ................................ ................................ .... 28 Introduction to KL 4 ................................ ................................ ................................ .. 29 Discovery of KL 4 ................................ ................................ ............................... 29 Structure and Function of KL 4 ................................ ................................ .......... 30 2 BACKGROUND FOR TECHNIQU ES AND METHODOLOGIES ............................ 33 Introduction ................................ ................................ ................................ ............. 33 Peptide Synthesis ................................ ................................ ................................ ... 33 Site directed Spin Labeling ................................ ................................ ..................... 34 Circular Dichroism Spectr oscopy ................................ ................................ ............ 37 Continuous Wave Electron Paramagnetic Resonance Spectroscopy .................... 40 Introduction ................................ ................................ ................................ ....... 40 Nitroxide Spectral Line Shapes ................................ ................................ ........ 43 Line Shape Data Analysi s ................................ ................................ ................ 45 Power Saturation CW EPR Spectroscopy ................................ .............................. 48 Introduction ................................ ................................ ................................ ....... 48 Statistical Analysis ................................ ................................ ............................ 50 Applications ................................ ................................ ................................ ...... 52

PAGE 7

7 3 OPTIMIZATION FOR CONTINUOUS WAVE ELECTRON PARAMAGNETIC RESONANCE STUDIES ................................ ................................ ......................... 53 Introduction ................................ ................................ ................................ ............. 53 Materials and Methods ................................ ................................ ............................ 53 Materials ................................ ................................ ................................ ........... 53 Methods ................................ ................................ ................................ ............ 54 Preparation of lipid/peptide samples ................................ .......................... 54 CW EPR spectroscopy ................................ ................................ .............. 54 Power saturation experiments ................................ ................................ .... 56 Lipid Composition ................................ ................................ ................................ ... 57 Results & Discussion ................................ ................................ .............................. 57 Effect of Negatively Charged Phosphatidylglycerol Lipids ................................ 57 Power Saturation Optimization ................................ ................................ ......... 65 NiAA Buffer Optimization ................................ ................................ .................. 67 Temperature Selection ................................ ................................ ..................... 68 Conclusions ................................ ................................ ................................ ...... 69 4 CONTINUOUS WAVE ELECTRON PARAMAGNETIC RESONANCE STUDIES OF KL 4 /LIPID INTERACTIONS ................................ ................................ .............. 70 Introduction ................................ ................................ ................................ ............. 70 Materials & Methods ................................ ................................ ............................... 70 Materials ................................ ................................ ................................ ........... 70 Methods ................................ ................................ ................................ ............ 71 Spin labeling of KL 4 cysteine mutants ................................ ........................ 71 Preparation of lipid/peptide samples ................................ .......................... 71 Circular dichroism spectroscopy ................................ ................................ 72 CW EPR spectroscopy ................................ ................................ .............. 72 Results & Discussion ................................ ................................ .............................. 73 Introduction ................................ ................................ ................................ ....... 73 KL 4 Effects o n Lipid Dynamics ................................ ................................ ......... 74 Mobility Parameters ................................ ................................ .......................... 77 Lipid Mobility Conclusions ................................ ................................ ................ 83 KL 4 Dynamics ................................ ................................ ................................ ... 84 Conclusions ................................ ................................ ................................ ............ 89 5 POWER SATURATION STUDIES ON KL 4 ................................ ............................. 91 Introduction ................................ ................................ ................................ ............. 91 Materials & Methods ................................ ................................ ............................... 91 Materials ................................ ................................ ................................ ........... 91 Methods ................................ ................................ ................................ ............ 92 Spin labeli ng of KL 4 cysteine mutants ................................ ........................ 92 Preparation of lipid/peptide samples ................................ .......................... 92 CW EPR spectroscopy ................................ ................................ .............. 93 Power saturation experiments ................................ ................................ .... 93

PAGE 8

8 Results & Discussion ................................ ................................ .............................. 94 Introduction ................................ ................................ ................................ ....... 94 Effect of KL 4 on Acyl Chain Accessibility ................................ .......................... 94 Effect of K L 4 on Spin label Depth ................................ ................................ ..... 98 Insertion Depth of KL 4 ................................ ................................ .................... 1 00 Conclusions ................................ ................................ ................................ .......... 103 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 104 Conclusions ................................ ................................ ................................ .......... 104 Future Directions ................................ ................................ ................................ .. 106 CW EPR Studies on SP B C terminus ................................ ........................... 106 Pulsed EPR on KL 4 by Electron Spin Echo Envelope Modulation (ESEEM) .. 106 CW EPR KL 4 Studies in Different Lipid Systems ................................ ............ 107 LIST OF REFERENCES ................................ ................................ ............................. 108 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 120

PAGE 9

9 LIST OF TABLES Table page 1 1 Pulmonary surfactant make up by weight %. ................................ ..................... 21 1 2 Di seases related to imprope rly functioning p ulmonary surfactant ....................... 23 1 3 Amino acid sequence of SP B termini and SP B analogs. ................................ 30 2 1 Common microwave bands and frequencies used in CW EP R ......................... 43 3 1 Standard CW EPR para meters ................................ ................................ ......... 56 3 2 Comparison of P 1/2 values measured with NiAA at pH 7.4 and pH 6.5 .............. 67 4 1 Standard parameters used for circular dichroism experiments. .......................... 72

PAGE 10

10 LIST OF FIGURES Figure page 1 1 closed sphere of gas ....................... 19 1 2 h the size of the sphere varies ................................ ................................ ................................ ................. 20 1 3 Schematic of the processes occurring at the highly dynamic fluid i nterface in the alveolus ................................ ................................ ................................ ....... 22 1 4 Five leading causes of neonatal mo rtality ................................ .......................... 24 2 1 Typical amino acid analysis ( AAA) mass spectrometry ................................ ..... 34 2 2 The site directed spin labeling scheme iodoacetamido PROXYL (IAP) ................................ ................................ ........... 35 2 3 CW EPR spectra for various scenario s ................................ ............................. 36 2 4 Struct ure of 1 palmitoyl 2 stearoyl (7 doxyl) sn glycero 3 phosphocholine and 1,2 dipalmitoyl sn glycero 3 phosp ho(tempo)choline ................................ 37 2 5 E lliptical polarized light ................................ ................................ ...................... 38 2 6 Sample circular dichroism sp ectra ................................ ................................ ...... 39 2 7 An energy diagram for a free electron in an applie d magnetic field .................... 42 2 8 An energy diagram for a syste m with a free electron being split into three allowed energy transitions due to the hyperfine interaction with a nitrogen nucleus. ................................ ................................ ................................ .............. 42 2 9 Dependence of EPR line shape on motion ................................ ......................... 44 2 10 Common spin labels utilized in SDSL ................................ ................................ 44 2 11 Spectral representation of de termining three c ommon mobility parameters ...... 45 2 12 Two spectra indicating the parameters used to calculate an order para meter .. 46 3 1 Tempera ture control set up ................................ ................................ ............... 55 3 2 CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 3:1 POPC:POPG lipid vesicles. ................................ ................. 58 3 3 CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 3:1 POPC:POPG lipid vesicles. ................................ ................. 59

PAGE 11

11 3 4 CW EPR spectra collected at 45 C und er nitrogen for 12 doxyl PC incorporated in 3:1 POPC:POPG lipid vesicles. ................................ ................. 59 3 5 CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles. ................................ ................. 60 3 6 CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles. ................................ ................. 60 3 7 CW EPR spectra collected at 45 C under nitrogen for 12 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles. ................................ ................. 61 3 8 Order parameter calculations for 5 doxyl spin label incorporated i nto 4: 1 POPC:POPG and 3:1 POPC:POPG lipid mixtures. ................................ ............ 62 3 9 Order parameter calculations for 7 doxyl spin label inco rporated into 4:1 POPC:POPG and 3:1 POPC:POPG lipid mixtures. ................................ ............ 62 3 10 pp calculations for 5 doxyl incorporated i nto 4:1 POPC:POPG and 3:1 POPC:POPG lipid mixtures. ................................ ................................ ............... 63 3 11 pp calculations for 7 doxyl in corporated i nto 4:1 POPC:POPG and 3:1 POPC:POPG lipid mixtures. ................................ ................................ ............... 63 3 12 pp calculations for 12 doxyl incorporated i nto 4:1 POPC:POPG and 3:1 POPC:POPG lipid mixtures. ................................ ................................ ............... 64 3 13 Power saturation values for NiEDDA and NiAA as a function of KL 4 concentra tion ................................ ................................ ................................ ...... 66 4 1 CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles with varying amounts of KL 4 .... 74 4 2 CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles with varying amounts of KL 4 .... 75 4 3 CW EPR spectra collected at 45 C under nitrogen for 12 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles with varying amounts of KL 4 .... 75 4 4 CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 4:1 DPPC:POPG lipid vesicles with varying amounts of KL 4 .... 76 4 5 CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 4:1 DPPC:POPG lipid vesicles with varying amounts of KL 4 .... 76 4 6 CW EPR spectra co llected at 45 C under nitrogen for 12 doxyl PC incorporated in 4:1 DPPC:POPG lipid vesicles with varying amounts of KL 4 .... 77

PAGE 12

12 4 7 pp plotted as a function of KL 4 concentration for 5 doxyl PC incorporated in 4:1 DPPC:POPG a nd 4:1 POPC:POPG lipid vesicles. ................................ ....... 78 4 8 pp plotted as a function of KL 4 concentration for 7 doxyl PC incorporated in 4:1 DPPC:POPG and 4:1 POPC:POPG lipid vesicles. ................................ ....... 78 4 9 pp plotted as a function of KL 4 concentration for 12 doxyl PC incorporated in 4:1 DPPC:POPG and 4:1 POPC:PO PG lipid vesicles. ................................ ... 79 4 10 pp graphed as a percent change to illustrate differences for 5 doxyl PC incorporated in 4:1 DPPC:POPG and 4:1 POPC:POPG lipid vesicles. ................................ ................................ ................ 80 4 11 pp graphed as a percent change to illustrate differences for 7 doxyl PC incorporated in 4:1 DPPC:POPG and 4:1 POPC:POPG lipid vesicles. ................................ ................................ ................ 80 4 12 pp graphed as a percent change to illustrate differences for 12 doxyl PC incorporated in 4:1 DPPC:POPG and 4:1 POPC:POPG lipid vesicles. ................................ ................................ ................ 81 4 13 Order parameters for 5 & 7 doxyl PC incorp orated in 4:1 DPPC:POPG and 4:1 POPC:POPG lipid vesicles as a function of KL 4 concentration. .................... 82 4 14 This graph show s the rati o of the central resonance line and the low field line for 4:1 DPPC:POPG a nd 4:1 POPC:POPG ................................ ....................... 83 4 15 The helical wheel representations of KL 4 ................................ ........................... 85 4 16 CD spectra for KL 4 ................................ ................................ ............................. 86 4 17 Spectra for all eight spin labeled peptid es ................................ .......................... 86 4 18 pp mea surements for KL 4 IAP individually spin labeled ................................ 87 5 1 1/2 (oxygen) plotted as a function of KL 4 mole perc ent ................................ ................................ ............................ 95 5 2 1/2 (NiAA) plotted as a function of KL 4 mole percent ................................ ................................ ............................... 95 5 3 1 /2 (oxygen) plotted as a function of KL 4 mole percent ................................ ................................ .............. 97 5 4 1/2 (NiAA) plotted as a function of KL 4 mole percent ................................ ................................ .............. 98 5 5 4 concentration .......................... 99

PAGE 13

13 5 6 4 conc en tration ................................ ................................ ................................ ... 100 5 7 4 IAP plotted as a function of spi n label position .... 101 5 8 Models of KL 4 pa rtition ing into DPPC rich and POPC rich regions. ................. 103

PAGE 14

14 LIST OF ABBREVIATION S AAA Amino acid analysis AMP Antimicrobial peptides ARDS Adult respiratory distress syndrome CD Circular dichroism CDC Center for disease control CrX Chrom ium oxylate CW Continuous wave DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPC Dipalmitoylphosphatidylcholine DTT Dithiothreitol EPR Electron paramagnetic resonance ER Endoplasmic reticulum ESEEM Electron spin echo envelope modulation FDA Food and d rug administration FTIR Fourier transform infrared KOH Potassium hydroxide LEU Leucine LUV Large unilamellar vesicle LYS Lysine IAP Iodoacetamido PROXYL IASL 4 (2 Iodoacetamido) TEMPO ICBR Interdisciplinary center for biotechnology IRDS Infant respiratory distress syndrome

PAGE 15

15 MeOH Methanol MLV Multilamellar vesicles MSL 4 Maleimido TEMPO MTSL (1 oxyl 2,2,5,5 tetramethyl pyrroline 3 methyl)methanethiosulfonate NaCl Sodium chloride NiAA Nickel (II) acetylacetonate NiEDDA Nickel (II) ethylene diamine diacetate NMR Nuclear magnetic resonance PAP Pulmonary alveolar proteinosis PC Phosphocholine PG Phosphoglycerol POPC 1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine POPG 1 palmitoyl 2 oleoyl sn glycero 3 phosphoglycerol PSPC 1 palmitoyl 2 steroyl 3 phosphocholine RDS Respiratory distress syndrome RP HPLC Reverse phase high performance liquid chromatography SDSL Site di rected spin labeling SP Surfactant protein ssNMR Solid state nuclear magnetic resonance TCEP T ris(2 carboxyethyl)phosphine TEM Transmission electron microscopy TFA T rifluoroacetic acid TFE Tetrafluoroethylene UV Ultra violet

PAGE 16

16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy AN ELECTRON PARAMAGNETIC RESONANCE STUDY OF SURFACTANT PROTEIN B MIMIC KL 4 By Austin Lisle Turner May 2012 Chair: Gail Fanucci Major: Chemistry KL 4 is a 21 amino acid peptide used to mimic the C terminus of lung surfactant protein B, a protein known to lower the surface tension in the highly dynamic alveoli. Understanding how KL 4 interacts with lipid vesicles of varying composition will provide insight into potential treatment for diseases such as respiratory distress syndrome. Recent 31 P and 2 H NMR studies have shown that KL 4 binds differently to POPC:POPG and DPPC:POPG multilamellar vesicles, with t he latter being found at elevated levels in lung surfactants. The current study uses electron paramagnetic resonance spectroscopy (EPR) and a technique called power saturation to study the effects of KL 4 binding to lipid bilayers both at the lipid and pept ide level. Power saturation can be used to determine a change in the accessibility of the spin label to molecular oxygen in the bilayer interior and to NiAA, an aqueous soluble nickel complex. Using information gathered from these experiments we will provi de insights into the depth and orientation of the peptide within different bilayer systems.

PAGE 17

17 CHAPTER 1 INTRODUCTION TO PULM ONARY SURFACTANTS Surfactants Surfactants are defined as compounds that lower the surface tension of a liquid, and the term is an a cronym of surface active agents Usually amphiphilic compounds such as lipids, surfactants are able to interact both with hydrophilic and hydrophobic molecules, giving them a wide variety of functions. Surfactant molecules typically migrate to the air/wa ter surface, as this is an interface between hydrophilic and hydrophobic environments. Typically thought of as detergents, wetting agents, dispersants, foaming agents, or emulsifiers, their function is to lower the interfacial tension between two states i n contact whether it be liquid liquid, liquid solid, or liquid gas. Self assembly into a wide variety of aggregate forms such as micelles, bilayers, or multilamellar vesicles, give surfactants of interest well defined properties and functions. Self aggre gation is controlled by the hydrophobic effect; in particular, lipids assemble into bilayers to minimize exposure of the hydrophobic tails to aqueous environments resulting in vesicular structures. The type of vesicle formed is dictated by the shape and c hemical structure of the lipids themselves. Lipids with large head group cross sections compared to their acyl chain cross section allow for a higher degree of curvature, ultimately resulting in micelle formation. Lipids such as phospholipids, in which t he cross section from top to bottom of the lipid is relatively constant, are commonly seen in more planar bilayers like those that allow for compartmentalization of the many organelles within cells. The tremendous importance of surfactants in life cannot be overstated as they allow for life as people know it to exist. Greater detail as

PAGE 18

18 to their behavior will be given in the sections that follow, giving particular focus to a specialized group of surfactants termed pulmonary surfactants. Properties of Surf actants Surfactants are found throughout living organisms including some of the most complex, dynamic systems of the human body such as mammalian lungs and are also used in a variety of simple household products such as laundry detergent. Many classes of surfactants exist, but they share one common feature which is their ability to reduce surface tension between immiscible states. To better understand this, a look at how surface tension arises needs to be understood, and an example of this is to look at a droplet of water upon a table and the curvature of the water droplet instead of a flat, evenly dispersed appearance. This phenomenon occurs because within the droplet each molecule is being pulled equally in all directions resulting in a net zero force. However, at the surface of the droplet there is not a cancellation of forces due to the absence of molecules on one side. Therefore, molecules on the surface are pulled inwards creating an internal pressure, forcing the liquid surface to contract to mini mize area hence, the curvature. In other words, this surface tension characterizes the shape of the droplet and can be altered by introduction of a surfactant. The surfactant adsorbs to the interface (liquid gas in this scenario) and reduces the interf acial tension between these two states, therefore, reducing the curvature seen in the droplet. This is a very basic description, but it can be used when looking at more complex systems such as pulmonary surfactant. Surfactants are of utmost importance an d a further look into how they function at a physical level and at a biological level will follow.

PAGE 19

19 General Mechanism of Surfactant Function To get a better understanding of surfactant function, consideration of the forces via interfacial regions to the shape of the surface or wall. Visually, a closed sphere of gas is contained within a uniform solution of water. Figure 1 1 illustrates this and can be described by LaPlac 1, wh thickness is presumed to be negligible. Figure 1 surrounded by a uniform aqueous environment. (1 1) In this scenario, the surface tension, which is perpendicular to the press ure, carries a value depending on the internal pressure and the external pressure pushing in on the sphere. This system is, therefore, in equilibrium and will remain so until a change is exerted upon it, which is the case in many dynamic systems. If this model system was used to interpret a relevant biological system, (e.g. the alveoli in the lung) there would

PAGE 20

20 be changes occurring to the system, for example inhalation and exhalation, in which the variables of the equation must change to keep the system at equilibrium. Figure 1 2 illustrates such a case in which the alveoli are changing in size and are modeled as perfect spheres in which the pressure differential, Figure 1 varies, such as the alveoli in the lung. From Equation 1 1 there is an inverse relationship between the radius of the alveolus and the surface tension. If the two differing size radii were to have the same surface tension, the alveoli with the smaller radius would experience a greater force inward, expelling its contents to larger alveoli eventually leading to collapse. This is the physics behind the need for surfactant in reducing the surface tension upon exhalation in the pulmonary system to prevent alveolar collapse. Introduction to Pulmonary Surfactants Pulmonary surfactant is a vital, lipid rich fluid found throughout the air fluid interface of alveoli. It is comprised largely of surface active lipids, mainly phospholipids and in particular dipalmitoylphosphatidylcholine (DPPC), along with a small mole percentage of hydrophilic and hydrophobic proteins. Pulmonary surfactan t is produced

PAGE 21

21 in type II epithelium cells lining the alveoli beginning towards the end of gestation. It facilitates air expansion, prevents collapse, and allows for easy re expansion of the lungs by drastically reducing alveolar surface tension in a highly dynamic, organized process [1 5]. In addition, it is believed to play a critical role in providing host defense against infection [5 9]. Relative lipid and protein percentages collected via bronchial lavage are shown in Table 1 1 [10 14]. Table 1 1 Pu lmonary surfactant make up by weight %. Lipid / Protein Relative weight % 1 ,2 dipalmitoyl sn glycero 3 phosphocholine (DPPC) 40 Phosphoglycerols (PG) 8 Unsaturated phosphocholines (PC) 25 Cholesterol 4 Surfactant Proteins (SP A, B, C, D) 7 Plasma Proteins 3 Other Lipids 7 Fatty Acids 6 These values can vary greatly due to age, environmental factors, dis ease, and whether in exhalation or inhalation [7, 8, 15, 16]. For example, surfactant production increases drastically at birth when the alveoli transition from fluid filled with negligible surface tension, to gas filled with substantial surface tension [ 9]. This is one of the underlying issues with preterm infant survival rates, as the lung has not had adequate time to develop and produce appropriate amounts of surfactant [17, 18]. Many factors go into producing sufficient amounts of surfactant includin g the secretion from Type II alveolar cells. This process is not fully understood but is believed to occur in both a continuous manner and via regulated pathways [7, 9, 19 22]. Packaging of the surfactant is done within T ype II cells in large concentric bilayers called lamellar bodies. From here the lamellar bodies are secreted into the fluid phase surrounding the alveoli and unravel into highly structured tubular myelin. Although not required, the tubular myelin assists in the

PAGE 22

22 rapid adsorption of a mon olayer of surfactant at the air fluid interface [1, 2, 18 26]. Furthermore, since the system is in continuous flux, the surfactant lipids have a turnover rate with a half life of just five to ten hours, emphasizing the need for proper surfactant catabolis m and recycling [9, 27, 28]. This is performed by alveolar macrophages and the type II cells which secrete the surfactant [28]. Figure 1 3 illustrates the process occurring near the alveoli air fluid interface. In the following sections a closer look wi ll be given to the individual components that make up pulmonary surfactant in addition to problems that may arise from deficiencies. Figure 1 3. Schematic of the processes occurring at the highly dynamic fluid interface in the alveolus, which i ncludes a high rate of lipid turnover (every 5 to 10 hours). Surfactant proteins are shown interacting with lipids in an arbitrary placement. Pulmonary Surfactant Deficiency Diseases Pulmonary surfactant plays such a vital role in proper lung functio n that any disturbance in production can drastically alter breathing capabilities. This leads to a wide range of diseases as illustrated in Table 1 2 [18, 29, 30]. Air Fluid

PAGE 23

23 Table 1 2 Several diseases related to improperly functioning pulmonary surfactant [18, 29 30] Disease Possible Cause Adult Respiratory Distress Syndrome (ARDS) Infection is most common Infant Respiratory Distress S yndrome (IRDS) Insufficient surfactant production Pulm onary Alveolar Proteinosis (P AP) Surfactant accumulation Congenital Surfactant Deficiency Genetic defects Pneumonia Inactivation / defi ciencies Asthma Inactivation / deficiencies Unfortunately, due to the complexity and highly dynamic nature of the alveoli, treatment for many lung diseases is difficult and can last a lifetime. Also, since the lung is the singular source of oxygen required by human cellular processes, affliction with some of these disorders can be fatal. This was the case with infant respiratory distress syndrome (IRDS) until the first successful tr ial of surfactant replacement therapy was developed by Fujiwara et al in 1980 [31, 32]. In the past thirty years since this landmark trial, RDS treatment has expanded from exogenous surfactant treatments to entirely synthetic options and will be discusse d in more detail in the following section [32 42]. Respiratory Distress Syndrome Respiratory distress syndrome affects people of all ages, is caused by a wide range of factors, and leads to life threatening illness. Typically classified into two categorie s, acute respiratory distress syndrome (ARDS) and infant respiratory distress syndrome (IRDS), each is marked with an increase in alveolar surface tension resulting A RDS this is caused by factors such as: lack of surface active compounds, changes in lipid composition, altering of surfactant protein composition, and inhibition of proper surfactant function by plasma protein leakage, among others. These factors can be

PAGE 24

24 b rought on by infection, massive trauma, pneumonia, pancreatitis, and multiple blood transfusions, in addition to environmental factors like toxic gas inhalation [43 46]. In the case of IRDS, the most common factor leading to the disorder is lack of surfac e active compounds. This occurs because the lung is not needed during gestation; therefore, it is one of the last organs to fully develop. When an infant is born preterm, the lungs have not had enough time to produce sufficient levels of pulmonary surfac tant and require treatment to allow for normal lung function [1, 18, 32, 47]. In both ARDS and IRDS, the treatment of choice is administering either extracted lung surfactant from an exogenous source or, preferably, a synthetic version in which the immuno genic response is reduces. The surfactant is introduced via a breathing tube directly into the lungs [18, 31, 45, 47]. Since the start of this type of treatment in the early 1980 s, a significant reduction in infant mortality rates has been shown [48 51]. Increasing interest has been generated in the study of RDS due to the statistics illustrated in Figure 1 4. Figure 1 4. Five leading causes of neonatal mortality, with RDS coming in fourth (left) and resulting in longest average hospital stay (rig ht).

PAGE 25

25 Surfactant Lipids There are multiple components that make up functioning lung surfactant as shown in Table 1 1 and they can be grouped into two categories, surfactant lipids and surfactant proteins [10, 18]. The latter will be discussed in the follo wing section as they play a vital role interacting with the lipids to allow proper lung function. Of the lipids found in lung extract, the high content of DPPC is especially unique when compared to other biological systems. This is because DPPC has two f ully saturated acyl chains, giving it a high melting temperature of 41 C. This places it above a physiological temperature of 37 C, making it rather unique among the lipid family. Having DPPC at the air fluid interface in alveoli makes sense though, as its fully saturated acyl chains allows it to be tightly packed or highly compressed and give it the ability to drastically reduce surface tension. When Clements et al. began looking at lung surfactant extracts back in 1961 ; they compared them with pure D PPC to understand its importance [52]. It was determined by Langmuir Blodgett trough measurements, and later confirmed by a captive bubble surface tensiometer, that the reduction of surface tension seen in lung surfactant extract was nearly identical to t hat of pure DPPC [52 55]. This is unusual given the composition shown in Table 1 1 where DPPC only makes up about 40 % of lung surfactant. Current theories, however, hypothesize that there is an enrichment process occurring at the air fluid monolayer in which DPPC is preferentially adsorbed [1]. This is very plausible as the average lipid composition is determined by lavage procedures involving a saline solution administered into the lungs [10, 18]. This process reveals an average concentration of lipid s present; however, there are many concerns with obtaining surfactant in such a manner. The introduction may disrupt the proper biophysical organization of the lipids, therefore generating surfactant form that may not

PAGE 26

26 exist in vivo. In addition, the comp osition obtained cannot discriminate from the microenvironment of the surfactant at the air fluid interface and the bulk lipid composition found in lamellar bodies or other large scale structures. Therefore, these numbers give a starting point to use for model studies but should be taken with skepticism as to the exact air fluid interface lipid composition. Another notable observation was by Hallman et al in 1975 when elevated levels of phosphatidylglycerol (PG) in healthy lung extract were seen when com pared to premature lung extract [56 58]. It was determined that PG contributed to surfactant spreading and along with DPPC were two key lipids involved in proper lung function [59]. These two lipids will go on to play critical roles in the advancement of RDS treatments and will b e extensively studied in later Chapters 3 5 of this work. Interactions with surfactant proteins are the next big step in understanding the molecular mechanisms underlying the function of surfactant at the dynamic air fluid interf ace. Surfactant Proteins Surfactant proteins make up about 10 % by weight of the bronchoalveolar lavage system [1, 6, 60 64]. Surfactant proteins are divided into two categories, hydrophilic surfactant proteins A and D and hydrophobic surfactant proteins B and C. The hydrophobic proteins B and C have been shown to promote rapid adsorption of lipids to the air fluid interface and are critical in proper lung function [65 69]. The hydrophilic proteins A and D aid SP B and SP C in promoting adsorption and play an important role in lung defense [60 64, 70]. Surfactant proteins B and C are small, hydrophobic proteins with a highly conserved primary sequence and are ne cessary for surfactant function in vivo [32, 71].

PAGE 27

27 An important function of both SP B and SP C is their ability to form monolayers by themselves or as mixtures with DPPC, in addition to promoting re adsorption of materials from collapsed, DPPC containing m onolayers [65, 67]. Their ability to perform these functions leads to their critical importance as shown in SP B knockout studies in which animals either had significantly decreased lung compliance or did not survive after delivery [72 74]. An important finding from these studies is th at SP A, SP C, SP D, and phosphatidyl choline concentrations remained normal, leading to a conclusion of the critical importance of SP B [75]. Further detail on SP B will be discussed in the following section. SP C gene kno ckout mice were generated and had normal lung function with only minor abnormalities [76]. Surfactant protein A is the most abundant protein in pulmonary surfactant and, like SP D, is related to a family of water soluble proteins called collectins, whic h contain both collagenous regions and C type lectin domains [77]. The C type lectin domains, also called a carbohydrate recognition domain (CRD), bind to specific complex carbohydrates of microbes allowing for the innate immune response and elimination [ 78]. In addition, SP A binds to DPPC, while SP D has been shown to bind to phosphatidylinositol (PI). Both are present in lung surfactant [77]. SP A also interacts with alveolar T ype II cells, implicating it in proper formation of the highly structure t ubular myelin [77, 79]. SP A and SP D play key roles in the innate immune system of the lung by allowing for immediate antibody independent host defense [32, 60 64, 70]. Knockout studies of SP A and SP D genes have shown retention of normal lung function in addition to achieving adequate minimal surface tensions of compressed films when compared to wild type [79]. However, due to their role in host defense,

PAGE 28

28 infections such as streptococci and pseudomonas commonly occurred in the knockout mice and they w ere more prone to die [80, 81]. Findings from these studies leads to an understanding of the importance of surfactant proteins to uptake and release of surfactant lipids from Type II epithelial cell; they however, do not provide a detailed understanding of their role in lowering surface tension [1, 82]. Surfactant Protein B and Analogs Lung surfactant protein B is an extremely hydrophobic homodimer with two 79 81 amino acid disulfide linked subunits. Each monomer contains an additional six cysteines used in intramolecular disulfide bonding [24]. These disulfide bonds, along with its high hydrophobicity, make protein purification difficult. Synthetic, peptide based lung surfactant replacements for treatment of RDS have shown promise and would reduce the purification difficulties and immunologic risks associated with exogenous animal derived lung surfactant [83, 84]. Chemically synthesizing the entire dimerized SP B is a daunting task so efforts have focused on producing truncated proteins and synthetic analogs of the N and C termini (SP B 1 25 SP B 59 80 and KL 4 ) as well as a fusion construct of the N and C termini of SP B, termed Mini B, which are less hydrophobic and lack the disulfide bridges of SP B [42, 83 86]. This has proven successf ul since much of the activity of SP B in lipid organization and dynamics have been attributed to the 20 25 amino acids on the N and C termini [83, 84, 87]. As with SP B, the N and C termini form helices when exposed to a lipid environment; however, th eir individual roles are not well understood. Although both ends of SP B have shown considerable surface activity by themselves, similar activity to native SP B has only been achieved by a construct of

PAGE 29

29 both the N and C termini [88 93]. A fully syntheti c mimic of SP B has achieved great interest due to its ease of synthesis, low cost, and elimination of exogenous immunological drawbacks. Cochrane and Revak began much of the synthetic mimic research in the early 1990s with KL 4 which has achieved great s uccess in FDA trials [94]. A more detailed look at KL 4 follows in the next section. Introduction to KL 4 Discovery of KL 4 KL 4 is an entirely synthetic mimic of the SP B C terminus which, much like SP B and its analogs, forms helices in lipid environments, but its role in lipid trafficking is often unclear [42, 95]. It was developed by Cochrane and Revak, along with several other synthetic peptides to resemble the hydrophobic and hydrophilic domains of the SP B C terminus [94]. The 21 mer KL 4 peptide, KLLL LKLLLLKLLLLKLLLLK demonstrates great clinical success in treatment of RDS and is currently available on the pharmaceutical market as one of the active components in the first generation, completely synthetic, lung surfactant replacement product Surfactant (Lucinactant). [92, 96 103]. Table 1 3 compares the amino acid sequence of SP B C terminus, KL 4 and Mini B (construct of N and C termini). Although KL 4 is a promising treatment for RDS, molecular level information on how it modulates surface tension in alveolar compartments is lacking. The increased effectiveness of KL 4 when compared to other commercially available formulations in addition to its vast difference in amino acid sequence suggest that understanding the way it affects the molecular and bi ophysical properties of the lipids is of great importance [104].

PAGE 30

30 Table 1 3 Amino acid sequence of SP B termini and SP B analogs. Peptide Peptide Sequence SP B 1 25 FPIPLPYCWLCRALIKRIQAMIPK SP B 59 80 DTLLGRLMPQLVCRLVLRCSMD KL 4 KLLLLKLLLLKLLLLKLLLLK Mini B CWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS Structure and Function of KL 4 As with the terminal ends of SP B, KL 4 has been found to adopt a helical conformation in a lipid environment, as shown with circular dichroism and Fourier transform infra red spectroscopy (FTIR) [42, 95, 105]. There have, however, been competing theories as to whether the helix partitions into the bilayer, either in the plane of the bilayer or in a transmembrane orientation. To further elucidate the structure, function, and orientation of KL 4 in lipid bilayers, the following lipid systems are typically used: fully monounsaturated POPC:POPG (3:1) and DPPC:POPG (4:1). The latter contains the fully saturated DPPC lipid and is being used in the therapeutic formulation, Lucina ctant. Models for KL 4 partitioning into lipid bilayers have fallen into two categories; a transmembrane helix and a helix lying in the plane of the bilayers. The 21 residue length of KL 4 allows it to adopt a transmembrane helix; however, it would bury 2 3 charged lysine side chains in the hydrophobic interior. A high percentage of leucine side chains and secondary structure could overcome this barrier, but previous studies using similar amino acid ratios suggest that a transmembrane orientation is only l ikely when the lysines are pos itioned closer to the N and C termini [106]. Nonetheless, recent studies have shown contradicting evidence in which a transcription translation assay found KL 4 capable of overcoming the energetic barrier and crossing the mem brane [107], and another 2 H solid state NMR study in which KL 4 was shown to adopt a helix lying in the plane of the bilayer [86]. The former

PAGE 31

31 studies compared the ap p app Prediction Server v1.0, http://www.cbr.su.se/Dgpred/) for insertion in endoplasmic reticulum (ER) membranes of KL 4 and the naturally occurring sequences, SP B 59 79 and SP C 13 35 which KL 4 is most closely mimicking. The se studies predict that SP C 13 35 is transmembrane ( 4.35 kcal/mol), an transmembrane orientation of SP B 59 79 would be unfavorable (+3.12 kcal/mol), and KL 4 a p p closer to SP C 13 35 ( 2.14 kcal/mol) [25, 107 109]. Their experiments to assay insert ion used a transcription translation assay with integration of KL 4 into Escherichia coli inner membrane protein leader peptidase (Lep), which is translated in ER derived microsomal membranes and assayed for glycosylation using proteinase K to determine whe ther the peptide is transmembrane. This assay, however, has two major drawbacks when used with KL 4 First, the addition of the Lep protein may influence the secondary structure of KL 4 in the lipid bilayer. Secondly, KL 4 is known to alter lipid dynamics and trafficking, therefore, altering the overall integrity of app helix conformation which may not be the case, as other studies using ssNMR have suggested KL 4 adopts a structure with a lower helical pitch in DPPC:POPG and POPC:POPG membranes [42]. With these structures its hydrophobic moment would be increased 3 4 kcal/mol, making transmembrane insertion much less favorable [86]. The latter study involving 2 H solid state NMR sug gests that KL 4 adopts a helix lying in the plane of the bilayers due to difference seen in the dynamics of leucine side chains which, if transmembrane, would have similar dynamics [86]. These studies, however, looked only at two sides of the helix instead of a complete turn around the predicted helix. Studies using FTIR have also led to conflicting models of the structure and

PAGE 32

32 orientation of KL 4 In early work using DPPC: DPPG (7:3), KL 4 was found to be helical and spanning the bilayer in a transmembrane c onfirmation [95]. Later FTIR work in either DPPC or DPPC:DPPG (7:3) mixtures have shown that KL 4 lies along the surface helix [110]. However, once again these assays assume that KL 4 helix which may not be the case. To further complicate this system, it has been suggested that KL 4 adopts a helix in the plane of the bilayer while also penetrating the bilayer to different extents depending on whether the fully saturated DPPC lipid is used. This was shown indirectly with 2 H and 31 P NMR studies of deuterated lipids and suggested t hat KL 4 partitions further into DPPC:POPG (4:1) than POPC:POPG (3:1) vesicles [86]. Understanding the orientation within the lipid bilayer will provide insights into its functional properties and assist in further development in effective respiratory distr ess syndro me treatments. In C hapters 4 6 we will optimize the lipid mixture compositions, power saturation parameters, and buffer pH. In addition, we will determine the orientation of KL 4 and depth profiles in two lipid systems, DPPC:POPG (4:1) and POPC: POPG (4:1) vesicles.

PAGE 33

33 CHAPTER 2 BACKGROUND FOR TECHN IQUES AND METHODOLOG IES Introduction Methods such as automated solid phase peptide synthesis, site directed spin labeling (SDSL), circular dichroism spectroscopy (CD), continuous wave electron paramagnet ic spectroscopy (CW EPR), and CW power saturation were utilized in the work of this dissertation. General overviews for each of these methods are given in the following sections. Specific details related to my studies are also provided in subsequent mater ia l and methods sections of each C hapter. Peptide Synthesis The relatively small size of KL 4 KLLLLKLLLLKLLLLKLLLLK containing 21 amino acids, rendered it easily amendable to peptide synthesis. KL 4 was synthesized via automated solid phase peptide synth esis on a Wang resin at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida, using an Applied Biosystems ABI 430 peptide synthesizer operated by Dr. Alfred Chung [92, 111, 112]. The peptide was cleaved from the resi n with 90% trifluoroace tic acid (TFA)/ 5% triisopropyl silane/ 5% water and ether precipitated. Purification of the cleaved product was carried out by reverse phase high performance liquid chromatography (RP HPLC) using an acetonitrile/ water gradient cont aining 0.3 % TFA. Fractions corresponding to KL 4 were collected and purity and concentration was verified by amino acid analysis (AAA) at the Molecular Structure Facility at the University of California, Davis. This technique allows for a high level of a ccuracy in concentration and purity by using Edman degradation, fluorescent labeling, and analytical HPLC to determine the amino acid c oncentrations from the N to C terminus direction of small peptides (<30 amino

PAGE 34

34 acids) [113, 114]. In addition to nativ e KL 4 cysteine variants were synthesized. In that process, individual leucines were replaced by cysteine (C7 C10, C12 C15) purified and mass verified (m/z=2459) in the same manner as stated above. Figure 2 1 shows one of the amino acid analysis results for KL 4 Figure 2 1. Typical amino acid analysis (AAA) mass spectrometry used to determine the relative number of amino acids per peptide (right). A result of 16 leucines for every 5 lysines verifies native KL 4 Site directed Spin Labeling Site directed spin labeling (SDSL) is a technique that introduces a paramagnetic species at a specific site in a protein or other biological molecule, typically by chemical modification of a cysteine residue, and renders the molecule EPR active [115]. When use d with EPR, conformations, conformational changes, solvent accessibility, and bilayer depth profiles are among a few of the details that can be extracted from the specific labeled site making it a well suited technique to study membrane bound protein or pe ptides [116 123].

PAGE 35

35 Site directed spin labeling exploits the chemical reactivity of the cysteine residue to allow for attachment of various spin labels [117, 119, 124]. Typically a cysteine residue is introduced via site directed mutagenesis, in which the D NA is manipulated such that a codon for cysteine is positioned at a chosen point within the sequence [115, 119]. As KL 4 is readily available by automated solid phase peptide synthesis, changing a desired leucine to a cysteine is well suited for this techn ique. After KL 4 has been modified to include the cysteine, the peptide construct is further modified with a spin label. The label of choice for these studies is iodoacetamido PROXYL (IAP) because it forms a non reducible carbon sulfur bond and is shown in Figure 2 2. Figure 2 2. The site directed spin thiol with iodoacetamido PROXYL (IAP) used to form the non reversible carbon sulfur bond. The resulting modified cysteine residue is referred to as R1 and will be used in KL 4 studies described in this dissertation. The sensitivity of the EPR nitroxide spectral line shapes resulting from these various spin labels to local dynamics, conformational changes, and local secondary structural elements allows rese archers to monitor the spin label environment using CW EPR spectrometers (Figure 2 3) [117, 118, 125 127]. To better elucidate information from the spectral line shape the types of motion that are being detected by the SDSL EPR technique need to be unders tood. There are three types of motion to consider in interpreting SDSL results: the intrinsic motion of the nitroxide spin label, the backbone motion due to the flexibility with the region of the protein the label is attached, and the

PAGE 36

36 overall tumbling of the peptide or protein. Each of these can be altered experimentally by changing the spin label of choice, temperature, viscosity, or altering the overall size of the tumbling system [119]. A variety of semi quantitative line shape parameters will be used and discussed in the subsequent EPR section. Figure 2 3. CW EPR spectra for various scenarios: unrestricted motion on the EPR timescale (A), gradually more restricted motion (B & C), and a powder like spectra in which the spin label is almost stati c (D). In addition to studying the EPR spectra of SDSL proteins, another useful CW EPR technique that assists in elucidating information about membrane bound proteins is to introduce a spin label onto a lipid, either on the acyl chain as a doxyl nitroxide at varying positions or on the polar head group in the form of a TEMPO nitroxide (Figure 2 4) [117, 120, 128 130]. Spin labeled lipids compliment well with SDSL protein for EPR studies as an understanding of both the protein and lipid membrane environment s can be determined. Addition of a small amount of spin labeled lipid (1 mol %) to a lipid system of choice gives excellent sensitivity for CW EPR line shape analysis. In addition, it allows for additional techniques such as power saturation to be perfor med to determine a relative depth profile which will be discussed later. Using a low concentration of doxyl lipid minimizes perturbation of the lipid system and limits interaction between neighboring spins. Spin labeled lipids are also widely commercial ly available at purities

PAGE 37

37 of >99% with doxyl labels at positions 5, 7, 10, 12, 14, and 16 along the acyl chain of PSPC as well as a Tempo label attached directly to the head group of DPPC or POPC (Figure 2 4). In addition to these commercially available, s everal groups have synthesized additional spin labeled lipids [131, 132]. Figure 2 4. Structure of 1 palmitoyl 2 stearoyl (7 doxyl) sn glycero 3 phosphocholine (left) and 1,2 dipalmitoyl sn glycero 3 phospho(tempo)choline (right). Circular Dichroism Spectroscopy Circular dichroism (CD) is a technique that observes differences in absorption between left and right handed circularly polarized light as a function of wavelength. Most notably, this technique is used to study optically active molecules su ch as biological molecules [133, 134]. The secondary structures of such molecules impart sheet, and random coil content [135 137]. To understand the technique, circularly polarized light m ust be R and E L being the magnitudes of the electric field vectors of right and left circularly polarized light, respectively (Figure 2 that there is complete absorbance of the circularly polarized light i n one direction; hence, the light is circularly polarized and by definition, chiral. This contrasts with linearly polarized light in which there is no difference between E R and E L The chiral property of circularly polarized light allows it to interact with chiral molecules in a distinct way which in turn gives rise to its importance in studying biological molecules. When circularly polarized light passes through a protein sample, the

PAGE 38

38 difference in absorption of left and right circularly pol arized light gives rise to an ellipticity which can be measured to generate a wavelength dependent plot of differential absorption of circularly polarized light. Figure 2 5. Elliptical polarized light (purple) is composed of unequal contributions f rom right (red) and left (blue) circular polarized light. Figure adapted from Wikipedia. When circular dichroism is used to determine secondary structural elements in proteins or peptides, the far ultraviolet (UV) region monitored is 180 250 nm, where the peptide bond is absorbing the left and right handed circularly polarized light to different sheet, or random coil, a distinct CD spectrum will be obtained (Figure 2 6). In the case of membrane bound proteins in which lipid vesicles are used, scattering from the lipids occur in the region below 200 nm, so CD spectra are typically truncated to the 200 250 nm region [136, 138 140]. Changes in secondary structure can be eluci dated by comparing CD spectra as a function of sequence and environment and used to determine perturbations in the peptide or protein secondary structure. Circular dichroism instrumentation, however, does not account for important experimental factors in r ecording the raw data spectra, so calculations are needed to unify reported CD spectra.

PAGE 39

39 Figure 2 6. Sample c sheet (blue), and random coil (green). Figure adapted from Chapman. Raw data is typically 2 dmol 1 residue 1 ), as shown in Equation 2 r is the protein molecular weight, c i s the protein concentration (mg/mL), l is the cell path length, and N A is the number of amino acids. (2 1) Further factors during samp le preparation must also be considered when accurately measuring secondary structure of the protein sample. Purity of sample is of high importance as any non target proteins will alter the overall secondary structure ensemble leading to misinterpretation of data. In addition, with a focus on secondary structure, the protein must be properly folded in an ideal buffer of low concentration (< 5 mM) while maintaining structural integrity. Typical protein concentrations should be maintained around 0.5 mg/mL. Any additive to the sample should not absorb in the far UV region, with the exception of lipid vesicles mentioned earlier. When lipid vesicles

PAGE 40

40 are introduced they need to be accounted for in the background subtraction and extruded through nanoporous membr ane to reduce the size of the vesicles and reduce light scattering. The ease of CD spectroscopy makes it a popular tool when studying proteins and when altering proteins to enable other techniques such as SDSL EPR. There are drawbacks, however, to the interpretation of CD spectra as they give an overall view of the secondary structure rather than information on a specific region of interest. This means that although secondary structure is revealed, actual determination of a properly folded protein is n ot possible. Keeping this in mind, CD spectroscopy should be used strictly to show if a protein is changing its structure and never to determine if the protein is indeed properly folded into its functional form. When using SDSL it is always recommended t o do CD on the samples because it can show small changes in secondary structure due to the spin label perturbing the protein structure. In addition, CD is a non Continuous Wave Electron Par amagnetic Resonance Spectroscopy Introduction Electron paramagnetic resonance spectroscopy (EPR), also known as electron spin resonance (ESR), is a technique for studying chemical species that contain one or more unpaired electron. These paramagnetic spec ies include organic free radicals and inorganic transition metal complexes. Similarities exist between EPR and nuclear magnetic resonance (NMR) ; however, EPR looks at electronic spin transition while NMR looks at nuclear spin transitions. Although not as widely used as NMR, EPR has increased in use due to the introduction of SDSL which allows for its use in systems which are not naturally EPR active. In its simplest form, a free radical in solution, the

PAGE 41

41 electron has a magnetic moment and spin quantum num ber s = with spin states m s = 1/2. In the absence of an external magnetic field, these spin states are degenerate, simply meaning the energy of the two spin states, m s = +1/2 (antiparallel) and m s = 1/2 (parallel) are equal. When an external magnetic field is applied these states have different energies leading to transition energy between them known as the Zeeman Effect. The difference between these two energy levels is described by the Zeeman equation (Equation 2 2), where g e fa B is the Bohr magneton, and is the strength of the applied magnetic field. The Bohr magneton (9.274 x 10 24 J T 1 ), is a physical constant an d is expressed using Equation 2 3, where e is the ch puts it in terms of radians (1.054 x 10 34 e 31 kg). (2 2) (2 3) In practice, CW EPR is performed by keeping the frequency fixed as the magnetic field is swept with field modulation, therefore, this means the energy gap between m s = +1/2 and m s = 1/2 is changed until it matches the frequency of the microwave or resonance condition, at which point the unpaired electron can transition between the two spin states. Due to the Maxwell Boltzmann distribut ion there are typically more electrons in the lower energy state, which leads to a net absorption of energy. It is this absorption which is monitored and converted into an EPR spectrum. The energy diagram describing this is shown in Figure 2 7A, while th e simplest form of an EPR spectrum is shown in Figure 2 7B. While this is the case for the simplest free electron, more complicated systems are typically observed with more complex resulting spectra.

PAGE 42

42 For EPR being performed in conjunction with SDSL, in w hich a nitroxide spin label is used, the spin of the electron, s = interacts with the nuclear spin of the nearby nitrogen ( I = 1) via the hyperfine interaction. In this case, both the m s = +1/2 and m s = 1/2 energy levels are split into three hyperfine energy levels dues to the 2 I +1 splitting rule. The energy diagram for this case is shown in Figure 2 8A, in addition to a corresponding derivative of the absorption spectrum in Figure 2 8B. Figure 2 7. (A) An energy diagram for a free electron in an applied magnetic field. (B) A corresponding first derivative spectrum. Figure 2 8. (A) An energy diagram for a system with a free electron (ms = ) being split into three allowed energy transitions due to the hyperfine interaction with a nitrogen nucleus (mI = 1). (B) A derivative spectrum of a nitroxide spin label with the following energy diagram. As mention previously, in conventional CW EPR the frequency is held constant throughout the experiment while the magnetic field is swept; however, spec tra can be collected at different frequencies depending on the sample of interest. For most

PAGE 43

43 studies, including those presented in this dissertation, collection is done in the X band range where resonances occur at magnetic fields around 3480 Gauss (0.35 T ) and frequencies of about 9.75 GHz. Other common EPR frequencies can be found in Table 2 1. Table 2 1 Common microwave b ands and frequencies used in CW EPR. Band Frequency (GHz) B resonance (Gauss) X 9.75 3480 Q 34.0 12000 W 94.0 34000 Nitroxide Sp ectral Line S hapes As previously stated and shown in Figure 2 4 the nitroxide EPR spectral line shape is highly sensitive to motion of the spin label environment and changes dramatically with correlation time, or the time it takes for a molecule to rotate one radian on average. To reiterate this, Figure 2 9 shows EPR spectra, which span a range from low correlation times (top) to much higher correlation times (bottom). The dependence of correlation time on the mobility of the spin label can be broken down into three modes of motion [127, 142, 143]. The first is the internal correlation i ) is determined by the intrinsic local mobility of the spin label which varies with type of spin label. To an extent this is experimentally distinguished by choosi ng a wide variety of spin labels with different bulky head groups as well as connectivity differences. Several common spin labels are shown in Figure 2 10 wit h R1 corresponding to the boxes. R ) and is de termined by the overall rotation of the protein. Rotational correlation time is dependent on the size of the protein if it is solubilized or the size of the system if it is membrane bound in a lipid vesicle. Sample conditions play an important role in de R such as temperature, viscosity, and buffer make up.

PAGE 44

44 Figure 2 9. Dependence of EPR line shape on motion Figure 2 10. Common spin l abels utilized in SDSL. (A) (1 oxyl 2,2,5,5 t etr amethyl pyrroline 3 methyl) m ethanethiosulfonate (MTS L), (B) 3 (2 i odo acetamido) PROXYL (IAP), (C) 4 maleimido tempo (MSL), and (D) 4 (2 iodoacetamido) tempo B ) is determined by backbone fluctuations in the protein an d local dynamics affected by neighboring molecules. This mode changes when the spin label is located at different secondary and tertiary structure locations on a protein or when a protein undergoes a conformational change. Fast Intermediate Slow Rigid

PAGE 45

45 Line Shape Data A nalysis CW EP R line shapes can be analyzed in qualitative, semi quantitative, and quantitative manners. This is due to the mobility allowed for a given spin label reflects greatly on the overall breadth of a spectrum as well as specific spectral features. In terms of describe it in terms of breadth. Examples are shown in Figure 2 3 in which the spectrum goes from very narrow in Figure 2 3A to very broad in Figure 2 3D. A semi quantitative a nalysis of spectra can be done by measuring spectral parameters such as; peak to peak line width pp ); ratios between normalized intensities of the three resonance lines, most notably the central resonance line h(0) over the low field line h(1); the second moment (); and an order parameter (S) which compares the sp ectrum to that of a completely static and completely mobile spectrum, giving a value between 0 and 1. There are other mobility parameters used throughout the EPR community, but the focus of this dissertation will be on using these more commonly measured o nes. Figure 2 11 shows a graphical representation of the first three parameters and a more detailed explanation follows. The fourth parameter (S) involves a little more detail so it is shown in Figure 2 12. Figure 2 11. Spectral representation of de termining three common mobility parameters pp (B) normalized intensities and (C) second moment (). I r 0

PAGE 46

46 Figure 2 12. Two spectra indicating the parameters used to calculate an order parameter (S) as typically used in doxyl lipid mobility calcul ations. pp is defined as the distance, in Gauss, between the maximum and minimum of the central resonance in a first derivative pp will experience ei ther a narrowing or broadening which can easily be seen in Figure 2 9. A pp while a longer correlation time pp In addition to looking directly at the H pp a scaled mobility, M s pp measured by the spectral line widths of the most mobile and immobile proteins to compare data collected on different instruments. Equation 2 4 gives the formula for calculating M s i m exp pp from the most immobile, most mobile, and experimental EPR spectra, respectively. (2 4) Comparison of normalize d resonance peak intensities is another way of quantitatively comparing mobility of spin labels between two or more samples. This involves normalizing all derivative spectra to the same number of spins via double integration and comparing two of the three normalized EPR resonance intensities, low

PAGE 47

47 field I LF center field I CF or I HF Most commonly, the ratio of the center field and low field intensities (I CF /I LF ) is used to compare spin labeled samples. In the case of highly anisotropic motion, the line s hapes become broadened as shown in Figure 2 1, which in turn leads to a decrease in the normalized intensities of each resonance. On the other hand, a fast isotropic motion generates an EPR line shape with narrow peaks of high intensities and therefore an increase in I CF /I LF This is a useful tool to use i n conjunction pp since the combination of multiple semi quantitative parameters gives a better overall understanding of the spectra. Spectral second moment calculations are different from the other parameters mentioned because they analyze the ab sorption spectra rather than the derivative spectra to determine an overall spectral breadth. Calculating second moments can be challenging as it involves a precise baseline correction and a need to account for any asymmetry contained within each spectrum Typically, second moments are the common moment found in literature, but in theory further moments can be calculated as shown in Equation 2 5, where H 0 is the center field, H j H j 1 is the step size, H j is the field value for any point j, and y j is the i ntensity at point j. (2 5) The last parameter shown is the order parameter, S, which is used when determining mobility around a doxyl spin label of interest and is determined using Equation 2 6. In determining S, the parameters T and T are evaluated for spectra with reduced mobility of the doxyl spin label. This being the case, S is usually not used for spin labeled acyl chains deeper in bilayers since the mobility is high in the fluid hydrophobic interior of membranes. In eva luating S, the experimental calculations

PAGE 48

48 (Figure 2 12) are compared with the case of zero mobility provided by simulations as shown in Equation 2 6 [144]. The order parameter ranges between zero to one for isotropic mobility to zero mobility, respectively This gives another useful tool in comparing data between instruments and different sample preparations. ) (2 6) Equation 2 6. Order parameter (S) calculations use the T and T measured from experimental data and compare them to those calculated for the rigid limit spectrum of a doxyl spin label, T zz and T xx In addition, polarity differences are taken into account for each s Power Saturation CW EPR S pectroscopy Introduction Power saturation is a CW EPR technique that introduces a secondary paramagnetic collider into the sample to look at the distance between the collider and the spin label via Heisenb erg exchange interactions [116]. Power saturation allows for an indirect measure of a relative depth parameter in the lipid environment for doxyl spin labels on lipids as well as nitroxide spin labels on proteins within a membrane. This technique exploit s the polarity gradient within lipid bilayers and the resulting partitioning of hydrophobic and hydrophilic molecules to the lipid and aqueous phases, respectively. In lipid bilayers there is a hydrophobic region, made up of lipid acyl chains, and a hydro philic region, comprised of the polar head groups and the surrounding aqueous environment. The bilayer interior, however, is non uniform, and consists of gradients of both fluidity and polarity along the bilayer normal [120, 145, 146]. Small molecules ca n diffuse into the bilayer with a gradient depending on both concentration and polarity

PAGE 49

49 dependent standard chemical potential in the bilayer, where C i,m (x) is the concen tration of species I in the bilayer at a distance x from the interface, C i,w is the uniform i,m i,w are the 7) [116]. (2 7) Introduction of another paramagnetic species, other than the spin label, either in the aqueous phase, such as a soluble transition metal complex, or in the hydrophobic int erior, such as molecular oxygen, allows EPR to benefit from this gradient. The paramagnetic collider that has been introduced into the membrane system of interest is allowed to interact, via Heisenberg exchange, with other paramagnetic species around it, such as the spin label. The rate at which the Heisenberg exchange occurs is proportional to the collision rate and is expressed by Equation 2 8, where W ex is the Heisenberg exchange rate, p is the exchange probability, g is a steric factor, d is the colli sion diameter, D m (x) is the position dependent relative diffusion coefficient, and C m (x) is the position dependent concentration given in Equation 2 8 [116]. (2 8) The collision rate is, therefore, dependent upon the depth in the bilayer in addition to many other factors such as steric hindrance, diffusion constants, and concentrations along the bilayer normal. However, by using two different paramagn etic colliders with relatively similar sizes and opposite partitioning gradients, i.e. a transition metal complex and oxygen, and by taking the ratio of their exchange rates with a given spin label, many of these factors cancel and the ratio solely depends on the distance through

PAGE 50

50 the concentration gradient. Studying the exchange rates between the colliders and the spin label allow determination of a depth profile within the bilayer. The paramagnetic colliders chosen are fast relaxing so their effect on ni troxide spin labels is dominated by Heisenberg exchange, as previously stated, and produces changes in the spin lattice relaxation time (T 1 ) of the spin label proportional to the collision rate, W ex [147]. The 1/2 which will be described in detail shortly, is related to this exchange rate according to Equation 2 9, where T 2e is the electron spin spin relaxation time. (2 9) Equations 2 7, 2 8, and 2 9 suggest that the logarithm of the experimental quantities 1/2 10. (2 10) paramagnetic colliders regardless of depth, with no consequence of viscosity or steric constraints caused by the system in study. In addition, using a ratio of the two colliders, T 2 shape and, therefore, useful over many systems. An assumption to this technique is that the chemical potentials have simple monotonic depth dependence, which is typically the c ase when two similar sized colliders are used. Statistical A nalysis Power saturation was developed and tested on the protein system bacteriorhodopsin by Dr. Hubbell and has since been used on many other systems

PAGE 51

51 [116]. The technique is performed by collecting EPR spectra of samples placed in gas permeable TPX capillary tubes so they can be purged with either air or nitrogen depending on the study of interest. The first derivative peak to peak amplitude, A, of the central resonance (m I =0) is measure d and plotted as a function of microwave power, P, over an incident power range of 0.2 63 mW. The resultant curves are fit to Equation 2 11. (2 11) Where I is a scaling f actor, P 1/2 is the power at which the resonance amplitude is one resonance spin [148]. P 1/2 values are obtained under three conditions for all peptide/lipid samples: hydrated vesic les equilibrated under nitrogen gas, hydrated vesicles equilibrated under air (20% oxygen), and hydrated vesicles equilibrated with aqueous, soluble 10 mM NiAA (or other aqueous soluble collider such as NiEDDA) 1/2 values for oxygen and NiAA are obtained by subtracting the P 1/2 value for nitrogen from the P 1/2 values of oxygen and NiAA. The depth parameter, 12. (2 12) Equation 2 13. (2 13)

PAGE 52

52 The values for NiAA were calculated b y substituting P 1/2 (NiAA) for P 1/2 (Oxygen) in the different laboratories regardless of the instrument or resonator used. Applications As previously mentioned, a depth profi le to determine partitioning depth within the bilayer can be achieved via power saturation [116]. This allows a wide range of applications such as; conformational changes of a membrane protein upon substrate binding, relative location of specific amino ac ids within a bilayer, and orientation of a protein in a bilayer (transmembrane, tilted, or in the plane of the bilayer) [116 119, 121 values experimentally determined at different SDSL posit ions along a membrane bound protein of interest [116]. A relative depth of amino acids can then be used to determine orientation and partitioning depth in the bilayer. This makes it a powerful tool for membrane bound proteins, which are notorious for bei ng difficult to structurally characterize in lipid bilayer environments [116, 131].

PAGE 53

53 CHAPTER 3 OPTIMIZATION FOR CON TINUOUS WAVE ELECTRO N PARAMAGNETIC RESONANCE STUDIES Introduction As described in Chapter 1 studies of surfactant protein B and its analogs are commonly done in DPPC:POPG (4:1) and POPC:POPG (3:1) lipid mixtures. DPPC:POPG (4:1) is commonly used in FDA approved treatments such as Survanta while POPC:POPG (3:1) is a lipid system mimicking cell membranes which is commonly employed to probe cat ionic, amphipathic protein interactions such as antimicrobial peptides (AMPs) with lipids [150 152]. Lipid phases of these compositions can likely be found in localized areas of alveoli during normal breathing cycles [153]. In C hapter 3 the content of PO PG between POPC:POPG (3:1) and POPC:POPG (4:1) and its effect on CW EPR will be discussed and explained. In addition, optimization of parameters such as temperature and choice of power saturation paramagn etic colliders for use in C hapters 4 and 5 will be discussed. Materials and Methods Materials POPC, DPPC, POPG, and n doxyl PSPC were purchased as chloroform solutions from Avanti Polar Lipids (Alabaster, AL) and quantified by phosphate analysis (Bioassay Systems, Hayward, CA). Iodoacetamido PROXYL spin l abel (IAP) was purchased from Sigma and used as received. Unless otherwise stated, all other reagents were purchased from Fisher Scientific (Hampton, NH) and used as received. KL 4 KLLLLKLLLLKLLLLKLLLLK was synthesized via solid phase peptide synthesis ( ICBR Facility, UF), purified by RP HPLC, and verified by mass spectrometry

PAGE 54

54 (m/z=2469). Peptide was dissolved in methanol and analyzed by amino acid analysis to determine concentration (Molecular Structure Facility, UC Davis). Methods Preparation of lipi d/peptide samples Lipid mixtures were prepared by mixing appropriate volumes of stock lipid chloroform solutions. For samples containing peptide, the peptide was added as a methanol solution to the lipid mixture in chloroform. Organic solvents were evapo rated using dry nitrogen; the lipid films were re suspended in warm cyclohexane (~45 C), flash frozen and lyophilized. For each combination of lipids and peptide, two separate samples were prepared. The first was rehydrated with 140 mM NaCl, 10 mM Bis T ris buffer, pH 6.5, and the second was rehydrated with 140 mM NaCl, 10 mM Bis Tris buffer, pH 6.5, containing 10 mM NiAA or 20 mM NiEDDA. The hydrated dispersions were subjected to 5 freeze thaw cycles to form MLVs and had a final 10 mM lipid concentratio n. Samples containing spin labe led lipid had 1 mol% of either d oxyl PC or tempo PC added relative to the unlabeled lipids. CW EPR spectroscopy CW EPR spectra were collected on a modified Bruker ER200 spectrometer (Billerica, MA) with an ER023M signal ch annel, an ER032M field control unit, and a loop gap resonator (Medical Advances, Milwaukee, WI). Spectra of samples containing spin labeled were recorded at 45 C using a 2 mW power level. Temperature was regulated by passing either air or nitrogen gas t hrough a copper coil in a recirculating bath (Thermo Scientific) containing 40% ethylene glycol. This setup is shown in Figure 3 1.

PAGE 55

55 Figure 3 1. Temperature control set up; A) thermocouple thermometer, B) quartz Dewar around the sample in the l oop gap resonator, C) copper coil submerged in recirculating bath, D) gas connected to quartz Dewar. Samples are stored in a 20 C freezer and allowed to thaw before use on the EPR. Lipid samples are then heated to above their melting temperatures and pl aced in the loop gap resonator with the preheated quartz Dewar and allowed to equilibrate at least 20 minutes prior to sample collection. CW E PR spectra were collected with one Gauss modulation amplitude and 100 or 125 Gauss sweep widths; the latter was u sed for spin labeled peptide. Additional spectra were collected at 20 Gauss sweep widths pp Each spectrum contains 1024 points with an approximate center field of 3460 Gauss. Spectra were collected and averaged betw een 2 75 scans with a frequency of 9.6 9.7 GHz. Table 3 1 shows a complete list of the typical parameters used in CW EPR experiments.

PAGE 56

56 Table 3 1 Standard CW EPR parameters used in this dissertation. Parameter Value Number of points 1024 Center field 3455 3465 G Number of scans 5 75 Sweep width 20 125 G Acquisition time 40.63 sec Frequency 9.5 9.7 GHz Power 0.25mW 20mW (29dB 10dB) Receiver gain 1x10 4 1x10 5 Modulation amplitude ~1 G Time constant 0.164 sec Receiver phase 100 105 deg Power saturation experiments Power saturation experiments were collected on the same modified Bruker ER200 spectrometer using gas permeable TPX capillary tubes as developed by Hubbell et al [116]. Saturation experime nts were collected at 25 C and 45 C for POPC:POPG for each experiment were made, one with and one without both 10 mM NiAA or 20 mM NiEDDA, and samples were purged for at leas t 20 minutes with air or nitrogen gas before collecting power saturation data. To ensure the samples were entirely purged with the gas, the intensity of the central resonance line was plotted as a function of time until no change in intensity was observed For each sample this was approximately 20 minutes. As mentioned in the previous section, intensity of the central resonance line using a 20 Gauss scan width was plotted as a function of microwave power in the range of 0.25 mW 20 mW. LabVIEW softwar e (National Instruments, Austin, TX) generously provided by Christian Altenbach and Wayne Hubbell (UCLA, Los Angeles, CA) was used for data recording and processing. Resultant power saturation curves were fit using Equation 2 11 and membrane depth was ana lyzed using Equation 2 12.

PAGE 57

57 Lipid Composition As previously mentioned, studies of surfactant proteins and their analogs are commonly done in DPPC:POPG (4:1) and POPC:POPG (3:1) systems [42, 85, 86, 105, 107, 153]. The latter lipid composition allows for c omparison to previous studies of cationic, amphipathic helix peptides which share similar characteristics to surfactant protein B [150 152]. While this allows for a reference to previously studied lipid/peptide systems, it does raise the question of how d ifferences in negatively charged POPG content in DPPC:POPG (4:1) and POPC:POPG (3:1) mixtures may lead to changes in the interactions of the lipid mixtures with positively charged lysines in KL 4 It has been postulated that the negatively charged POPG pla ys a critical role in SP B pulmonary lipid interaction and DPPC enrichment, and if this is true the positively charged KL 4 peptide should be no exception [153 159]. To better understand if EPR can detect differences in interactions of KL 4 with POPC:POPG a t a 3:1 versus a 4:1 ratio, CW EPR power saturation experiments using both ratios were performed. Results & Discussion Effect of Negatively Charged Phosphatidylglycerol L ipids The observation that pulmonary surfactant contains elevated levels of negative ly charged lipids when compared to most other biological systems, PG and PI, suggests they play a specific role in surfactant function. This has been demonstrated in multiple studies showing that mixing POPG or DPPG with DPPC enhances adsorption at the ai r/water interface during film formation while allowing for selective squeeze out of PG during lipid film compression [154, 156, 159]. In addition, these affects are not observed in the absence of SP B and SP C and show a much greater effect with SP B comp ared to SP C [155]. The ability of KL 4 to mimic SP B functions suggest that it too

PAGE 58

58 will be affected by PG content. The extent of its effect is not presently known so studies using lipid systems containing varying amounts of PG, like DPPC:POPG (4:1) and P OPC:POPG (3:1), have be en of recent concern. In C hapter 3 we use CW EPR mobility and power saturation studies to determine whether or not differences in POPG content affect the changes in lipid dynamics observed on addition of KL 4 Initial CW EPR spect ra were collected for both 3:1 and 4:1 POPC:POPG spin labeled lipids samples at varying concentrations of KL 4 to study the effects of binding on the lipid environment. All spectra collected for the two lipid systems, at concentrations of KL 4 varying from 0 to 3 mol percent, using lipids spin labeled at 5 7 12 doxyl positions, are shown in Figure 3 2 through 3 7. Figure 3 2. CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 3:1 POPC:POPG lipid vesicles. 2.0 mol % 1.0 mol % 0 .0 mol % 0.2 mol % 1.5 mol % 2.5 mol % 0 .5 mol % 3.0 mol % 10 Ga uss

PAGE 59

59 Figure 3 3. CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 3:1 POPC:POPG lipid vesicles. Figure 3 4. CW EPR spectra collected at 45 C under nitrogen for 12 doxyl PC incorporated in 3:1 POPC:POPG lipid vesicles. 2.0 mol % 1.0 mol % 0 .0 mol % 0.2 mol % 1.5 mol % 2.5 mol % 0 .5 mol % 3.0 mol % 10 Gauss 2.0 mol % 1.0 mol % 0 .0 mol % 0.2 mol % 1.5 mol % 2.5 mol % 0 .5 mol % 3.0 mol % 10 Gauss

PAGE 60

60 Figure 3 5. CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles. Figure 3 6. CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 4:1 POPC:POPG lipid v esicles. 2.0 mol % 1.0 mol % 0 .0 mol % 0.2 mol % 1.5 mol % 2.5 mol % 0 .5 mol % 3.0 mol % 10 Gauss 2.0 mol % 1.0 mol % 0 .0 mol % 0.2 mol % 1.5 mol % 2.5 mol % 0 .5 mol % 3.0 mol % 10 Gauss

PAGE 61

61 Figure 3 7. CW EPR spectra collected at 45 C under nitrogen for 12 doxyl PC incorporated in 4:1 POPC:POPG lipid vesicles. To further analyze the spectra, semi pp and order parameter (S) were used to compar e the two lipid systems. The se parameters are described in C hapter 2 Figure 2 11 and Equation 2 6. In Figures 3 8 and 3 9 order parameters are compared for 3:1 and 4:1 POPC:POPG lipid mixtures containing 5 and 7 doxyl spin labels with varying KL 4 concen trations. The order parameter (S) increases upon addition of KL 4 at both the 5 and 7 doxyl position in both lipid systems. This is indicative of KL 4 interacting with the spin label at both positions within the bilayer and decreasing its mobility. This i s expected because it is known that KL 4 binds to lipid bilayers containing PG [42, 86, 107]. Interestingly the rate of increase in order parameter is within error between 3:1 and 4:1 POPC:POPG samples, suggesting the small difference in PG content does no t alter the results seen by CW EPR at the 5 and 7 positions along the lipid acyl chain. As previously stated, order parameters can only be used for more immobilized spin labels and cannot be used for spin labels closer to the bilayer interior. To compa re pp was calculated for spectra of lipids 2.0 mol % 1.0 mol % 0 .0 mol % 0.2 mol % 1.5 mol % 2.5 mol % 0 .5 mol % 3.0 mol % 10 Gauss

PAGE 62

62 spin labeled at the 5 7 and 12 doxyl positions. Figures 3 10 through 3 12 compare the results at all three positions, in both lipid systems, and at varying K L 4 concentrations. Figure 3 8. Order parameter calculations for 5 doxyl spin label incorporated into 4:1 POPC:POPG (grey squares) and 3:1 POPC:POPG (open circles) lipid mixtures. Figure 3 9. Order parameter calculations for 7 doxyl spin la bel incorporated into 4:1 POPC:POPG (grey squares) and 3:1 POPC:POPG (open circles) lipid mixtures. 5 Doxyl 7 Doxyl

PAGE 63

63 Figure 3 pp calculations for 5 doxyl incorporated into 4:1 POPC:POPG (grey squares) and 3:1 POPC:POPG (open circles) lipid mixtures. Figure 3 pp calculations for 7 doxyl incorporated into 4:1 POPC:POPG (grey squares) and 3:1 POPC:PO PG (open circles) lipid mixtures. 5 Doxyl 7 Doxyl

PAGE 64

64 Figure 3 pp calculations for 12 doxyl incorporated into 4:1 POPC:POPG (grey squares) and 3:1 POPC:POPG (open circles) lipid mixtures. As seen with the order parameter plots in Figures 3 8 and 3 pp incr eases at the 5 and 7 doxyl positions upon addition of KL 4 indicating a restriction of motion due to the pp is seen for 3:1 and 4:1 POPC:POPG, giving further evidence that the small discrepancy in PG content does not affect the CW EPR line shape upon KL 4 addition. Furthermore, pp allows lipid acyl chain mobility to be determined semi quantitatively deeper in the bilayer at position 12. As shown in Figure 3 12, the chang pp upon peptide addition is negligible, suggesting KL 4 is not penetr ating deeply enough in either 3: 1 POPC:POPG nor 4:1 POPC:POPG lipid bilayers t o effect the mobility at the 12 position. Once again in both lipid systems the mobility parameter is w ithin error of the two lipid systems suggesting the charge difference between the two systems does not alter the doxyl spin label mobility. The CW EPR mobi lity studies presented in C hapter 3 show that the variability of negatively charged PG concentrat ion between 3:1 and 4:1 POPC:POPG gives no substantial change in EPR spectra, alleviating any concern about studies in which 3:1 POPC:POPG and 4:1 DPPC:PO PG are compared. For C hapters 4 and 5 equal ratios 12 Doxyl

PAGE 65

65 of zwitterionic and anionic (4:1) will be used in POPC:POPG and DPPC:POPG lipid mixtures. Power Saturation Optimization As stated in C hapter 2 power saturation is a technique which introduces a paramagnetic collider, either an aqueous soluble metal complex or hydrophobic oxygen, which interacts with a s pin label by Heisenberg exchange and measures relaxation to determine an accessibility parameter of the spin label with the collider. Molecular oxygen provides a convenient lipophilic paramagnetic collider as it is readily available from air and can be pu rged into the sample of interest with ease. As for the aqueous soluble paramagnetic collider there have been many choices that vary in price, aqueous solubility, and lipid solubility. A few choices commonly seen in power saturation studies are nickel (II ) acetylacetonate (NiAA), nickel (II) ethylenediamine diacetic acid (NiEDDA), and chromium oxylate (CrX) [116]. For the following studies, NiAA and NiEDDA will be compared in our lipid systems to determine advantages and disadvantages of each for fut ure use in power saturation studies. The aqueous solubility of NiEDDA is far greater to that of NiAA, allowing for concentrations of over 100 mM v ersus only 20 mM, respectively. This allows for greater interaction between the collider and a spin label that is exposed to the aqueous phase, resulting in an increased relaxation enhancement detected by power saturation. However, if for spin label sites buried within the hydrophobic phase, NiAA may be more advantageous because of its higher permeability wit hin lipid bilayers, leading to an increased interaction between the collider and the spin label. To determine which aqueous collider is best for our studies, CW EPR power saturation data was collected on spin labeled lipids samples containing either 10 mM NiAA or 20 mM NiEDDA. It

PAGE 66

66 should be noted that a higher concentration of NiEDDA was also tested and gave similar results to 20 mM NiEDDA. Figure 3 13 plots the effect of NiEDDA and NiAA on power saturation P 1/2 values in 4:1 POPC:POPG vesicles as a funct ion of KL 4 concentration and with varying spin labeled doxyl positions. Figure 3 1/2 ) for NiEDDA (left) and NiAA (right) as a function of KL 4 concentration with spin label reporters using 5 doxyl (open circles), 7 d oxyl (grey squares), and 12 doxyl (black triangles) spin labels. 1/2 for NiEDDA and NiAA plotted as a function of KL 4 show significant differences in accessibility between the two paramagnetic agents. The NiEDDA sam ples give P 1/2 values similar to those of nitrogen, indicating very little collision at each doxyl position with NiEDDA. In addition, the correlation 1/2 values and the doxyl depths do not show the logical trends seen with the NiAA. The 1/2 values should have a clear change with spin label position, or 5 doxyl > 7 doxyl > 12 doxyl. This is seen when using NiAA but not NiEDDA. For this reason NiAA was selected for future studies of spin label accessibility to an aqueous collider.

PAGE 67

67 NiAA Buffer Optimization The best suitable aqueous soluble collider for these studies is NiAA due to its favorable permeability into the bilayer allowing for higher rates of collision with all spin labels used. The next issue to be considered is an optimal b uffer to be used in power saturation studies with NiAA. Typically KL 4 is studied using a buffer consisting of 10 mM HEPES, 140 mM NaCl, pH 7.4 [42, 86]. This buffer, however, gave erroneous results for simple NiAA accessibility studies, and therefore oth er buffers were compared. Results comparing 10 mM HEPES, 140 mM NaCl, pH 7.4 and 10 mM Bis Tris, 140 mM NaCl, pH 6.5 are shown in Table 3 2. For these studies both POPC:POPG and DPPC:POPG vesicles were used with 10 mM NiAA. Table 3 2 Comparison of P 1/2 values measured with NiAA at pH 7.4 and pH 6.5 in two lipid systems. Spin label POPC:POPG (4:1) DPPC:POPG (4:1) pH 7.4 pH 6.5 pH 7.4 pH 6.5 5 Doxyl 19.0 0.4 19.3 0.2 19.4 0.3 19.6 0.3 7 Doxyl 21.5 0.7 16.8 0.5 22.4 0.5 16. 4 0.3 12 Doxyl 15.2 0.2 11.8 0.2 17.1 0.3 14.5 0.3 To focus specifically on the pH of the samples, each sample was made without KL 4 and with 150 mM salt to limit variability. For a buffer to be compatible with NiAA, a trend in P 1/2 values sho 1/2 values for 5 doxyl > 7 doxyl > 12 doxyl. This is clearly not the case at pH 7.4 where a trend of 7 doxyl > 5 doxyl > 12 doxyl is seen. This trend occurs in both POPC:POPG and DPPC:POPG vesicles a t pH 7.4, therefore raising the question about whether or not the

PAGE 68

68 charged PG head group is interacting in an unusual manner with the nickel complex or if the charge state of NiAA is being affected by the pH. Ultraviolet visible spectroscopy (UV Vis) studi es were performed on samples at varying pH to see if a shift in wavelength absorption could be seen indicating the NiAA was undergoing a charge state change. UV Vis spectroscopy did not indicate any change in absorbance as the pH was varied from 5.5 to 8. 5. A future study using transmission electron microscopy (TEM) to study changes in the morphology of the lipid vesicles containing NiAA at varying pH has been considered and could lead to a better understanding of the observed anomalous trend. Lowering t he pH to 6.5, however, alleviated this discrepancy in P 1/2 values for NiAA measured with spin labels at the 5 7 12 doxyl positions. Table 3 2 shows the expected trend 5 doxyl > 7 doxyl >12 doxyl for both lipid systems at pH 6.5 and is therefore used i n all future experiments. Temperature Selection When studying peptide partitioning into lipid environments knowing the melting temperature of the lipid system is of importance in order to regulate which phase is being studied. This especially is signific ant when using fully saturated lipids, like DPPC, because their high melting temperatures can cause them to be in a gel rather than liquid crystalline phase at room temperature. Careful consideration of experimental temperatures and sample preparation pro tocols should be made when preparing and analyzing saturated lipid systems. The three lipids used in this dissertation, POPC, POPG, and DPPC, have melting transition temperatures of 2 C, 2 C, and 41 C, respectively. This results in a melting tempera ture for our two systems, POPC:POPG (4:1) and DPPC:POPG (4:1), of 2 C and 37 C, respectively [85]. In addition, the large discrepancy between DPPC and POPG transition temperatures results in the need for

PAGE 69

69 careful sample preparation to prevent phase sepa ration of the lipid. This can be achieved by mixing in organic solvents at higher temperatures, followed by flash freezing and sublimation of each sample via lyophilization. To further complicate the issue, addition of KL 4 at high concentrations in DPPC: POPG shifts the transition temperature from 37 C to 39 C, suggesting a possible peptide induced phase separation [85]. To account for all these factors, 45 C was chosen for our studies as it ensures samples are above the melting temperature of all lipi ds and that any shift in transition temperature due to KL 4 addition will not affect the results. In addition, to allow easy comparison between POPC:POPG and DPPC:POPG, 45 C was used for all samples. This eliminates differences in mobility and solvent ac cessibility studies due to increased motion at higher temperatures. Conclusions To accurately study a system of interest, several parameters need to be optimized to give the most sensitive and reproducible results. In C hapter 3 the parameters of lipid composition, power saturation collider, buffer pH, and temperature were all studied to determine the most ideal conditions. These studies allow for an elimination of PG concentration variability, increased accuracy of depth profile measured by power satur ation, and comparison of EPR CW spectra between two different lipid systems. Future data collected and described in this dissertation will use these determined parameters in addition to future work.

PAGE 70

70 CHAPTER 4 CONTINUOUS WAVE ELEC TRON PARAMAGNETIC RE SONAN CE STUDIES OF KL 4 /LIPID INTERACTIONS Introduction C hapter 3 summarized the optimized conditions for CW EPR experiments which will be used in this Chapter and C hapter s 4 and 5 describing powe r saturation results. In C hapter 4 results from spin labeled l ipid and KL 4 CW EPR mobility studies will be reported and discussed. Mobility studies provided a means to understanding the local environment of the lipid spin label at varying positions along the acyl chain upon addition of KL 4 In addition, changes in mobility around the spin labeled KL 4 helix were tracked using eight KL 4 variants. Mobility studies can help to elucidate the orientation of KL 4 within the lipid bilayer in addition to helping understand differences between DP PC and POPC rich vesicles. B y studying changes occurring at different positions within KL 4 and along the acyl chain and by comparing this to the power saturation results in the C hapter 5 a depth profile of KL 4 in DPPC:POPG (4:1) and POPC:POPG (4:1) will be determined. Materials & Methods Materials POPC, DPPC, POPG, n doxyl PSPC were purchased as chloroform solutions from Avanti Polar Lipids (Alabaster, AL) and quantified by phosphate analysis (Bioassay Systems, H ayward, CA). Iodoacetamido PROXYL spin label (IAP) was purchased from Sigma and used as received. Unless otherwise stated, all other reagents were purchased from Fisher Scientific (Hampton, NH) and used as received. KL 4 KLLLLKLLLLKLLLLKLLLLK was synthesized via solid phase peptide synthesis (ICBR Facility, UF), purified by RP HPLC, and verified by mass spectrometry

PAGE 71

71 (m/z=2469). Peptide was dissolved in methanol and analyzed by amino acid analysis for concentration (Molecular Structure Facility, UC Davis). Cysteine variants of KL 4 in which individual leucines were replace d by cysteine, were also synthesized via solid phase peptide synthesis, purified, and mass verified (m/z=2459). Methods Spin labeling of KL 4 cysteine mutants Cysteine containing KL 4 was dissolved at a concentration of ~0.1 mM in MeOH, and the pH was adju sted to >7 with KOH. A five fold excess of tris(2 carboxyethyl)phosphine (TCEP) was added to keep the cysteine reduced. A solution of 50 mM IAP spin label in DMSO was added to achieve a 20 fold excess of spin label. After 4 5 hours at room temperature, the spin labeled peptide was purified via HPLC, and lyophilized fractions were brought up in MeOH. The final peptide concentration for each spin labeled sample was determined by either analytical HPLC or amino acid analysis (AAA). Preparation of lipid/p eptide samples Lipid mixtures were prepared by mixing appropriate volumes of stock lipid chloroform solutions. For samples containing peptide, the peptide was added as a methanol solution to the lipid mixture in chloroform. Organic solvents were evaporat ed using dry nitrogen; the lipid films were re suspended in cyclohexane, flash frozen and lyophilized. For each combination of lipids and peptide, two separate samples were prepared. The first was rehydrated with 140 mM NaCl, 10 mM Bis Tris buffer, pH 6. 5, and the second was rehydrated with 140 mM NaCl, 10 mM Bis Tris buffer, pH 6.5, containing 10 mM NiAA. The hydrated dispersions were subjected to 5 freeze thaw cycles to form MLVs and had a final lipid concentration of ~10 mM. Samples

PAGE 72

72 containing sp in labeled lipid had 1 mol% of d oxyl PC added relative to the native lipids during the organic solvent mixing step. Circular dichroism s pectroscopy CD spectra were acquired at 45 C on an Aviv Model 202 spectrometer using Hellma CD cuvettes with 1 cm path len gth with special thanks to Dr. Steve Hagan in the UF Physics department for assistance. Samples were prepared by hydrating lyophilized peptide lipid powders in 10 mM Bis Tris buffer, pH 6.5, with 140 mM NaCl, to achieve a final concentration of ~50 M KL 4 Samples were extruded through 100 nm filters (Avanti Polar Lipids, Alabaster, AL) to form LUVs. Typical parameters used for CD experiments are summarized in Table 4 1. Background scans of all buffers were collected and subtracted from the final averag ed spectra. Table 4 1. Standard parameters used for circular dichroism experiments. Parameter Value Experiment type Wavelength Bandwidt h 1 nm Temperature 45 C Wavelength range 260 190 nm Step gradient 1.0 nm Averaging time 3.0 sec Settling time 1.0 sec Multi scan wait 1.0 sec Scans 5 10 CW EPR s pectroscopy CW EPR spectra were collected on a modified Bruker ER200 spectrometer (Billerica, MA) with an ER023M signal channel, an ER032M field control unit, and a loop gap resonator (Medical Advances, Milwaukee, WI). Spectra of samples containing either spin labeled lipid or spin labeled KL 4 were recorded at 45 C using a 2 mW power

PAGE 73

73 level. The temperature was re gulated by passing nitrogen gas through a copper coil in a recirculating bath (Thermo Scientific) containing 40% ethylene glycol. Samples were stored in a 20 C freezer and allowed to thaw before use on the EPR. Lipid samples were then heated to above th eir melting temperatures and placed in the loop gap resonator within a preheated quartz Dewar and allowed to equilibrate at least 20 minutes prior to sample collection. CW EP R spectra were collected with one Gauss modulation amplitude and 100 or 125 Gauss sweep widths, the latter used for spin labeled peptide. Additional spectra were collected at 20 Gauss sweep widths for pp Each spectrum contained 1024 points with an approximate center field of 3460 Gauss. Spectra were collected and averaged for 2 75 scans at a frequency of 9.6 9.7 GHz. Table 3 1 shows a complete list of the typical parameters used in CW EPR. Results & Discussion Introduction As discussed in C hapter 1 the orientation and function of SP B and its analogs in lipid environments is not well understood. In particular, several studies have yielded conflicting results as to how KL 4 is oriented in a lipid bilayer and how it interacts with specific lipids. It is well known that KL 4 adopts a helix upon membrane binding as shown through previous FTIR and CD studies, in addit ion to CD studies presented in C hapter 4 [42, 95, 105]. The orientation of the helix, however, is believed to either span the bilayer in a transmembrane orientation [107], or lie in the plane of the bilayer [42, 86, 107]. Our CW EPR mobility studies will help to elucidate the true orientation and relative depth within the two lipid systems, 4:1 DPPC:POPG and 4:1 POPC:POPG, by studying changes in dynamics for both spin labeled lipids and peptide s. In addition,

PAGE 74

74 C hapter 5 will use CW power saturation EPR to correlate collision data with the m obil ity data presented in C hapter 4 KL 4 Effects on Lipid Dynamics The first study of CW EPR mobility was to example the changes in spin label mobility occurring at the 5 7 and 12 doxyl labeled lipid acyl chains upon adding increasing amounts of KL 4 into the two lipid systems of interest. Using the semi quanti tative parameters discussed in C hapter 2 an understanding of the local environment around each doxyl lipid in both lipid systems can be developed. Figure 4 1 through 4 6 displays the CW EPR spectra at all eight KL 4 concentrations, for all three doxyl positions and in both lipid systems. These spectra along with 20 Gauss spectra were used to determine semi quantitative parameters of spin labeled lipid mobility in C hapter 4 Figure 4 1. CW E PR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 4:1 POPC:POPG lipid vesi cles with varying amounts of KL 4 0 mol % 0 .2 mol % 0 .5 mol % 1. 0 mol % 1.5 mol % 2.0 mol % 2.5 mol % 3. 0 mol % 10 Gauss

PAGE 75

75 Figure 4 2. CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 4:1 POPC:POPG lip id vesi cles with varying amounts of KL 4 Figure 4 3. CW EPR spectra collected at 45 C under nitrogen for 12 doxyl PC incorporated in 4:1 POPC:POPG lipid vesi cles with varying amounts of KL 4 0 mol % 0 .2 mol % 0 .5 mol % 1. 0 mol % 1.5 mol % 2.0 mol % 2.5 mol % 3. 0 mol % 10 Gauss 0 mol % 0 .2 mol % 0 .5 mol % 1. 0 mol % 1.5 mol % 2.0 mol % 2.5 mol % 3. 0 mol % 10 Gauss

PAGE 76

76 Figure 4 4. CW EPR spectra collected at 45 C under nitrogen for 5 doxyl PC incorporated in 4:1 DPPC:POPG lipid vesi cles with varying amounts of KL 4 Figure 4 5. CW EPR spectra collected at 45 C under nitrogen for 7 doxyl PC incorporated in 4:1 DPPC:POPG lipid vesi cles with varying amounts of KL 4 0 mol % 0 .2 mol % 0 .5 mol % 1. 0 mol % 1.5 mol % 2.0 mol % 2.5 mol % 3. 0 mol % 10 Gauss 10 Gauss 0 mol % 0 .2 mol % 0 .5 mol % 1. 0 mol % 1.5 mol % 2.0 mol % 2.5 mol % 3. 0 mol %

PAGE 77

77 Figure 4 6. CW EPR spectra collected at 45 C under nitrogen for 12 doxyl PC incorporated in 4:1 DPPC:POPG lipid vesicles with varying amounts of KL 4 Looking qualitatively at the spectra in Figures 4 1 through 4 6, mobility increases as the position of the spin label increases (from 5 doxyl to 12 doxyl). This is due to the increased fluidity in the bilayer interior when compared to the membrane interface. Qualitative comparison of spectra between the two lipid systems and between spectra of lipids containing varying amounts of KL 4 is difficult, therefore a series of semi quantitative parameters were determined. Mobility Parameters As previously stated, there are several parameters which can be used to describe and quantita te changes in CW EPR spectra. The first parameter commonly pp which is determined as described in C hapter 2. Figures 4 7 through 4 pp values for spin labeled lipids between the two lipid systems at varying concentrations of KL 4 and at varying spin label dept hs within the bilayer. 10 Gauss 0 mol % 0 .2 mol % 0 .5 mol % 1. 0 mol % 1.5 mol % 2.0 mol % 2.5 mol % 3. 0 mol %

PAGE 78

78 Figure 4 pp plotted as a function of KL 4 concentration for 5 doxyl PC incorporated in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares) lipid vesicles. Figure 4 pp plotted as a function of KL 4 concentration for 7 doxyl PC incorporated in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares) lipid vesicles. 5 Doxyl 7 Doxyl

PAGE 79

79 Figure 4 pp plotted as a function of KL 4 concentration for 12 doxyl PC incorporated in 4:1 DPPC:POPG (blac k triangles) and 4:1 POPC:POPG (grey squares) lipid vesicles. Figures 4 7 and 4 8 show that as KL 4 concentration is increased, mobility is decreased at about the same rate for 5 and 7 doxyl labeled lipids in both lipid systems. This indicates that KL 4 is interacting with the spin label at these positions along the acyl chain upon membrane binding and doing so in similar manner between the two lipid pp trends for DPPC:POPG and POPC:POPG samples containing 12 doxyl labeled PSPC (Figure 4 9). For DPPC rich pp indicating an interaction between KL 4 and the 12 not seen in 4:1 POPC:POPG MLVs, suggesting the peptid e is not interacting as deeply in the bilayer as seen with 4:1 DPPC:POPG. To further illustrate the difference between pp is plotted as a function of KL 4 concentration and shown in Figures 4 10 through 4 12. 12 Doxyl

PAGE 80

80 Figure 4 pp graphed as a percent change to illustrate differences for 5 doxyl PC incorporated in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares) lipid vesicles. Figure 4 11. The mobility para pp graphed as a percent change to illustrate differences for 7 doxyl PC incorporated in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares) lipid vesicles. 5 Doxyl 7 Doxyl

PAGE 81

81 Figure 4 pp graphed as a percent chan ge to illustrate differences for 12 doxyl PC incorporated in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares) lipid vesicles. pp is a semi quantitative parameter which suggests that it alone does not entirely quantit ate mobility. For this reason several mobility parameters are typically used to better understand dynamics. For doxyl labeled lipids, another commonly used mobility parameter is the order para meter S, which is described in C hapter 2. To calculate the or der parameter, the axial hyperfine anisotropy must be discernible, which is the case for 5 and 7 doxyl labeled lipids but not for the highly isotropic 12 doxyl labeled lipids. Figure 4 13 shows the calculated order parameters for both 5 and 7 doxyl labe led lipids in both lipid systems as a function of peptide concentration. 12 Doxyl

PAGE 82

82 Figure 4 13. Order parameters for 5 & 7 doxyl PC incorporated in 4:1 DPPC:POPG (black) and 4:1 POPC:POPG (blue) lipid vesicles as a function of KL 4 concentration. As previo u pp measurements, the order parameters for 5 and 7 doxyl in both lipid systems increase with peptide concentration, indicating KL 4 is interacting with the spin labels at these depths. For both DPPC and POPC rich vesicles, the increase in S is similar upon KL 4 addition. For both semi pp and S, there is a similar trend of decreased mobility at the 5 and 7 doxyl positions in both lipid systems upon KL 4 binding. The fact that a difference was seen at the 12 position between the two systems and the inability to use S as a measure of mobility for 12 doxyl labeled lipid spectra, indicates another parameter needs to be studied to see if indeed there are differences at the 12 doxyl position. The final mobility parameter l ooked at is the ratio between the intensity of the first derivative central line with that of the low field line. A detailed description of this parameter is given in C hapter 2 and the results are shown in Figure 4 14. A more substantial increase in h(0) /h(1) is seen in DPPC rich vesicles when compared to POPC rich vesicles. To further illustrate this difference, the percent change in h(0)/h(1)

PAGE 83

83 was also graphed as a function of KL 4 concentration. This data correlates well with the pp in Fig ures 4 7 through 4 9, which suggest KL 4 penetrating further into DPPC rich vesicles and interacting with the 12 position of the lipid acyl chain. Figure 4 14. This graph shows the ratio of the central resonance line (h0) and the low field line (h1) for 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). The left graph shows the raw data and the right as a percentage change to illustrate the discrepancy betweeen DPPC:POPG and POPC:POPG. Lipid Mobility Conclusions Studying changes in mobility along the lipid acyl chain gives an understanding of how KL 4 pp S, and h(0)/h(1) suggest KL 4 displays similar effects on regions closer to the membrane interface in the two lipid systems as reported by 5 and 7 doxyl labeled lipids. However, it appears that KL 4 is interacting further in the bilayer at the 12 doxyl position in 4:1 DPPC:POPG lipids in contrast to 4:1 POPC:POPG lipids. In addition, the lack of an easily detected in teraction deep within the bilayer in 4:1 POPC:POPG lipids suggests a transmembrane orientation of KL 4 in this environment is unlikely These results correlate well with some recently published NMR data [42, 86, 107], specifically 2 H NMR

PAGE 84

84 data which looked at the dynamics of four deuterated leucine positions (3, 10, 12, and 19) [86]. The positions were chosen because two leucines (Leu3 and Leu19) were at the terminal ends while two leucines being in the center of the peptide (Leu10 and Leu12). For a transm embrane orientation, similarities in dynamics between Leu3 and Leu19 would be expected as would similarities between Leu10 and Leu12 because they would lie in the center of the bilayer. The 2 H NMR showed that Leu3 and Leu12 exhibited similar dynamics wh il e Leu10 and Leu 19 shared similar dynamics. In addition, Leu10 and Leu12 had dynamics that were significantly different which makes a transmembrane orientation highly unlikely. Additionally, further analysis of the leucine side chain dynamics suggested he lix helix packing due to aggregation was unlikely and the differences in dynamics seen are from a helix lying in the plane of the bilayer. The next section will look at changes in the dynamics of KL 4 via EPR to see if correlations can be made with lipid d ynamics EPR data and the previously published NMR studies. KL 4 Dynamics To properly study membrane bound proteins, analysis of both lipid and protein dynamics are needed. In the previous section, mobility studies were performed to understand changes in dynamics occurring along the lipid acyl chain upon adding increasing amounts of KL 4 In this section the spin label will be attached to the peptide via site directed spin labeling, as described in C hapter 2, and the peptide concentration will be held con stant at 2 mole percent. Eight spin label positions were chosen (C7 C10 and C12 C15) which allow for the study of two complete turns around the KL 4 helix. Helical wheel representations of KL 4 helix and as calculated by 13 C ssNMR peptide torsion a ngle studies by Dr. Long et al in POPC:POPG and DPPC:POPG vesicles are shown in Figure 4 15 [42, 86, 107].

PAGE 85

85 Figure 4 15. The helical wheel representations of KL 4 helix (A), in POPC:POPG vesicles (B), and DPPC:POPG (C) as predicted b y NMR studies. Orange amino acids represent positions which were individually spin labeled and blue amino acids represent positively charged lysines. Lysine at position 1 was left off of the NMR wheels due to its high flexibility. Arrows indicate the ne t hydrophobic moments resulting from the charged lysine side chains on the helix surface. Addition of a spin label to a peptide opens the possibility that the overall structure has been perturbed. A common way to study if a change has occurred in the o verall structure is using circular dichroism ( CD) as previously described in C hapter 2. Figure 4 16 shows CD for all eight spin labeled sites (C7 C10 & C12 C15) in both lipid systems in addition to native KL 4 and KL 4 in TFE and MeOH. No change in seconda ry structure from native KL 4 is seen upon addition of IAP spin label to any of the eight sites used. To compare differences in mobility at the different peptide positions on partitioning into the two lipid systems, CW EPR spectra were collected and analyze d. These spectra are shown in Figure 4 17 and were collected at 45 C with a 125 Gauss scan width. Each para meter measured is discussed in C hapter 2. To analyze each KL 4 IAP pp was calculated in the same manner as the previous section. Each spectrum was collected using 2 mol percent KL 4 pp was plotted as a function of spin pp is shown in Figure 4 18 along with the projected A B C

PAGE 86

86 helical wheels from KL 4 NMR data [42] for the peptide partitioning into POPC:POPG and DPPC:POPG lipid vesicles as shown in Figure 4 15. Figure 4 16. CD spectra for KL4 in TFE ( ---) and KL4 in MeOH (____) used as reference spectra. Overlaid spectra of C7 C10 & C12 C15 spin labeled KL4 IAP sites and native KL4 in POPC:POPG and DPPC:POPG (grayscale). Figure 4 17. Spectra for all eight spin labeled peptides incorporated in 4:1 POPC:POPG (blue) and 4:1 DPPC:POPG (black) lipid vesicles with 125 Gauss scan widths and 45 C are shown. KL 4 C7 KL 4 C8 KL 4 C 9 KL 4 C 10 KL 4 C12 KL 4 C 13 KL 4 C 14 KL 4 C15 POPC POPC DPPC DPPC

PAGE 87

87 Figure 4 pp measurements (center) for KL 4 IAP individually spin labeled at positions C7 C10 and C12 C15 and incorporated into 4:1 POPC:POPG (grey squares) and 4:1 DPPC:POPG (black triangles) lipid vesicles. Helical wheel representations for KL 4 redicted structure in POPC:POPG (left) and DPPC:POPG (right) lipid vesicles are shown for positioning reference NMR [42]. Studying the mobility of a spin pp allows for an understanding of the local environment at the spin labeled position. Dep ending on the orientation of KL 4 in pp ) would be expected. For example, if KL 4 were to span the bilayer in a transmembrane orientation, the N and C termini would be expected to have decreased mobility due to their position in the less mobile interface region of the lipid bilayer. On the other hand, if KL 4 were to lie in the plane of the bilayer an expected mobility pattern would repeat every four amino acids as one proceeds around the helix. This mean s that one side of the helix would be in a region of high fluidity (bilayer interior), while the opposite side has restricted mobility due to it being at the lipid interface. By choosing two groups of four consecutive amino acids, two full turns around th e peptide helix are being studied, as shown in Figure 4 18. The first four sites chosen, C7 C10, were used as they not only make a complete turn around the helix, but they also predicted to lie on opposite sides of KL 4 based on NMR

PAGE 88

88 studies [42, 86], givin g the largest possible discrepancy in mobility between each site (see Figure 4 18 helical wheels). In both POPC and DPPC rich vesicles, position 7 sits closest to the lipid head group while position 9 is deepest in the bilayer interior. In addition, posi tions 8 and 10 lie at similar depths about half way between positions 7 and pp In both POPC and DPPC vesicles the most restricted mobility is seen at position 7 and incre ases going to position 8 then 9 and finally decreasing slightly at position 10. The overall mobility pattern for this region of KL 4 pp for IAP at C 7 > C 10 > C 9. Consistent with a helix lying in the plane of the bilaye r the EPR data correlate well with the proposed helical model in Figure 4 18 and rules out the possibility of KL 4 spanning the bilayer. The values measure d in POPC and DPPC rich environments show similar trends in this region with a DPPC rich environment yielding pp values indicating slightly more restricted spin label mobility. This is to be expected since DPPC, being fully saturated, can more tightly pack allowing for a greater effect on peptide spin label mobility. The next set of spin labels monit ored were at positions C12 C15, which are the next set of leucines after the lysine following the first set of labels and they also make a full turn around the helix (Figure 4 18). For this set of spin labels the discrepancy predicted between where the po sitions lie in the bilayer is not as great as with the first set, giving similar depth positions for 12 and 15, which are closest to the bilayer interface, and 13 and 14 being similar but deeper in the bilayer. For both POPC and DPPC rich environments the pp values do not coincide as nicely with the helical wheel predictions as positions C7 C10. For DPPC vesicles (Figure 4 18 black

PAGE 89

89 pp values similar to expected from the helical wheel diagram, in which 1 2 and 15 are similar and 13 is more mobile. However, C14 pp value than any of the other seven positions. This is not to be expected for a helix in the plane of the bilayer with a helix pitch similar to those given in Figure 4 18. This i ntriguing result is also seen in the POPC rich environment as well in pp similar to that of C7, which is close to the bilayer interface. To better understand if these findings are due to sample preparation, power saturation studies wer e performed to see if similar results could be seen in solvent accessibility. Chapter 5 will go into the details of the power saturation results, but all indications pp mobility results are indeed real and may be caused by some pertur bation of KL 4 around the C14 positions. Two possibilities have been postulated as to the origin of these unexpected values; either KL 4 is slightly tilted in its orientation in the bilayer so as to not lie entirely parallel, to the bilayer planes of KL 4 co uld have a kink in its structure which might affect the relative mobility at specific spin labeled positions. C hapter 5 will look at the relative partitioning depth in the bilayer for each spin labeled position to add further insight into this issue. In addition, power saturation will address the possibility that KL 4 is affecting the bilayer by either thickening or thinning the bilayer. Conclusions The CW EPR mobility studies in this work are consistent with a model in which KL 4 lies in the plane of the lipid bilayer in both 4:1 POPC:POPG and 4:1 DPPC:POPG lipid environments. Spin labeled KL 4 mobility studies indicate a pattern consistent with a helix in the plane of the bilayer and rule out the possibility of KL 4 spanning the bilayer in a transmembrane orientation. Spin labeled lipid studies indicate that KL 4 is

PAGE 90

90 partitioning more deeply into DPPC:POPG vesicles compared to POPC:POPG vesicles. This is seen in differences in lipid mobility occurring at the 12 position on the lipid acyl chain upon addition of KL 4 This important difference may play a vital role in lipid trafficking and DPPC enrichment at the air f luid interface of the alveoli.

PAGE 91

91 CHAPTER 5 POWER SATURATION STU DIES ON KL 4 Introduction C hapter 4 summarized the mobility experiments performed t o understand the local environment around spin labels attached to specific lipid and peptide positions. Several relative mobility parameters were measured to give an understanding of how KL 4 is oriented in the bilayer and how it partitions differently int o 4:1 DPPC:POPG and 4:1 POPC:POPG environments. It was determined that KL 4 does not span the bilayer in a transmembrane orientation, but actually lies in the plane of the bilayer. As shown with previous NMR studies [42, 86], EPR mobility suggests that KL 4 partitions further into DPPC rich bilayers allowing for a possible mechanism of action in preferentially aff ecting DPPC dynamics. In C hapter 5 power saturation CW EPR will be employed to give an understanding of the relative depth of KL 4 in the two lip id systems by looking at the solvent accessibility of spin labels attached at specific lipid and peptide positions. The combination of CW EPR mobility studies and power saturation experiments gives a better understanding of KL 4 interacts these two lipid s ystems and allows for the development of a proposed KL 4 binding model. Materials & Methods Materials POPC, DPPC, POPG, n doxyl PSPC were purchased as chloroform solutions from Avanti Polar Lipids (Alabaster, AL) and quantified by phosphate analysis (Bio assay Systems, Ha yward, CA). Iodoacetamido PROXYL spin label (IAP) was purchased from Sigma and used as received. Unless otherwise stated, all other reagents were purchased from Fisher Scientific (Hampton, NH) and used as received.

PAGE 92

92 KL 4 KLLLLKLLLLKLLLLKLL LLK was synthesized via solid phase peptide synthesis (ICBR Facility, UF), purified by RP HPLC, and verified by mass spectrometry (m/z=2469). Peptide was dissolved in methanol and analyzed by amino acid analysis for concentration (Molecular Structure Faci lity, UC Davis). Cysteine variants of KL 4 in which individual leucines were replaced by cysteine were also synthesized via solid phase peptide synthesis, purified and mass verified (m/z=2459). Methods Spin labeling of KL 4 cysteine mutants Spin labelin g of KL 4 w as carried out as described in C hapter 4. The final peptide concentration for each spin labeled sample was determined by either analytical HPLC or amino acid analysis (AAA). Preparation of lipid/peptide samples Power saturation samples were pr epared as described in C hapter 4. For power saturation analysis, each sample was made as described previously but in duplicate to allow for one of the samples to contain a final concentration of 10 mM NiAA. One sample contained 140 mM NaCl, 10 mM Bis Tri s buffer, pH 6.5, and the second was rehydrated with 140 mM NaCl, 10 mM Bis Tris buffer, pH 6.5, containing 10 mM NiAA. This allows for the study of all three power saturation experiments; with nitrogen, oxygen, or NiAA. The hydrated dispersions were sub jected to 5 freeze thaw cycles to form MLVs and had a final lipid concentration of ~10 mM. Samples containing sp in labeled lipid had 1 mol% of d oxyl PC added relative to the native lipids and spin labeled peptide samples contained a constant 2 mole perce nt of KL 4 IAP for each experiment.

PAGE 93

93 CW EPR s pectroscopy Continuous wave EPR experiments were collected as described in C hapter 3. For power saturation curves a microwave power range of 0.25 mW to 25 mW was used to measure peak to peak intensities for eac h sample. Samples were purged and temperature was equilibrated by passing either air or nitrogen gas through a copper coil in a recirculating bath (Thermo Scientific) containing 40% ethylene glycol for 20 30 minutes prior to measurements. Initial spectra of 100 125 Gauss sweep width were collected to ensure samples were correctly prepared. Power saturation spectra were collected at 20 Gauss sweep widths centered on the central resonance line to more accurately determine the intensity of the peak. Po wer saturation experiments Power saturation experiments were collected on the same modified Bruker ER200 spectrometer using gas permeable TPX capillary tubes as developed by Hubbell et al [116]. Saturation experiments were collected at 45 C for both PO PC:POPG and each experiment were made, with or without 10 mM NiAA and purged for at least 20 minutes with air or nitrogen gas. To ensure the samples were entirely purged with the gas, the intensity of the central resonance line was plotted as a function of time until no change in intensity was observed. For each sample this was approximately 20 minutes. As mentioned in the previous section, intensities of the central resonance l ine at 20 Gauss scan widths, were plotted as a function of microwave power in the range of 0.25 mW 25 mW. LabVIEW software (National Instruments, Austin, TX) was used for data recording and generously provided by Christian Altenbach and Wayne Hubbell (U CLA,

PAGE 94

94 Los Angeles, CA). Resultant curves were fit using Equation 2 11 and analyzed using Equation 2 12. Results & Discussion Introduction Power saturation is a technique developed by Hubbell et al and is used to develop a depth parameter of the system of interest by studying the solvent accessibility of a spin label with its local environment. The proce dure is described in detail in C hapter 2 and will be used here to better understand the partitioning of KL 4 in both 4:1 DPPC:POPG and 4:1 POPC:POPG. The indications from previous experiments [42, 8 6] and the mobility studies in C hapter 4 which suggest that KL 4 partitions differently in DPPC bilayers, indicates that developing a depth profile for KL 4 using power saturation in these two systems will greatly advance our understanding of the partitioning. In addition, power saturation can aid in the understanding of the mobility studies by differentiating between a decrease in mobility of the spin label due to interaction with KL 4 or change due to a change in the bilayer thickness. The following sections will discuss the results from the spin label lipid and spin label peptide power saturation studies and compa re them to results from Chapter 4 and work of other groups/studies. Effect of KL 4 on Acyl Chain Acc essibility As previously discussed in C hapter 4, CW EPR mobility studies indicate KL 4 is partitioning deeper in DPPC and interacting with the 12 position on the lipids acyl chain. This interaction was not seen in POPC vesicles, as only a change in mobili ty was noticed at both the 5 and 7 doxyl positions. Power saturation will show if these mobility changes also correspond to changes in solvent accessibility of the spin label lipid, albeit aqueous NiAA or hydrophobic oxygen. To determine this, samples w ere prepared as

PAGE 95

95 mentioned in Chapter 5 methods section with varying amounts of KL 4 The accessibility 1/2 is plotted as a function of KL 4 concentration for oxygen and NiAA and is shown in Figures 5 1 and 5 2. An in depth description of all power satu ration parameters are given in C hapter 2. Figure 5 1. Power satu 1/2 (o xygen) plotted as a function of KL 4 mole percent. Samples contain either 5 doxyl (left), 7 doxyl (center), or 12 doxyl (right) in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). Figure 5 2. 1/2 (NiAA) plotted as a function of KL 4 mole percent. Samples contain either 5 doxyl (left), 7 doxyl (center), or 12 doxyl (right) in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). The accessi 1/2 is a measure of the spin labels interaction via Heisenberg exchange with the paramagnetic collider, either oxygen or NiAA, and 5 Dox yl 7 Doxyl 12 Doxyl 5 Doxyl 7 Doxyl 12 Doxyl

PAGE 96

96 therefore is a measure of the accessibility of the spin label and that collider. Figure 5 1 1 /2 for oxygen at the 5 7 and 12 doxyl positions in both lipid systems. 1/2 values increase as the spin label is moved further down the acyl chain (moving from left to right in Figure 5 1) which is due to oxygen being lipophilic, therefore, havin g a higher concentration and an increase probability of collision with the doxyl spin label. The opposite is seen in Figure 5 2 in which NiAA is most concentrated 1/2 value at the 5 doxyl position. Lo oking at differences in trends upon KL 4 addition at each position between the two lipid systems gives an understanding of changes in the local environment of the spin label. Figure 5 1 (left) shows a similar decrease in oxygen accessibility between the tw o lipid systems suggesting both a displacement of oxygen at the 5 doxyl position because of KL 4 binding and a decrease of spin label and oxygen collisions due to the reduced mobility of the spin label. The same trend is seen at the 7 doxyl position, Figur e 5 1 (center), which correlates well with the previous mobility studies which found similar interactions of KL 4 with both lipid systems at these positions. Deviation once again, occurs at the 12 doxyl position which shows very little change in oxygen acc essibility for 4:1 POPC:POPG and a substantial decrease for 4:1 DPPC:POPG. As with the mobility 1/2 values for oxygen suggest KL 4 penetrating further and interacting at the 12 doxyl position in DPPC rich vesicles but not in POPC rich vesicles. Th 1/2 (NiAA), is shown in Figure 5 2 and is plotted in the same manner as Figure 5 1. At the 5 doxyl position there is a decrease in NiAA accessibility upon KL 4 binding suggesting the interaction of KL 4 at that s pin label position. The trend is similar between the two lipid systems, however, there

PAGE 97

97 is a deviation at the higher KL 4 concentrations in which the accessibility of NiAA in POPC rich vesicles increases slightly whereas DPPC rich it continues to decrease. This may be caused by KL 4 interacting closer to the bilayer interface in POPC when compared to DPPC and altering the partitioning of NiAA around the lipid head groups in such a manner as to increase the collisions of NiAA and the 5 doxyl spin label. This deviation, however, is not seen at the 7 doxyl position and the expected similar 1/2 1/2 (NiAA) at the 12 doxyl position again shows the biggest deviation between the two systems. Like all the previou s data has suggested, KL 4 interacts further in the bilayer of DPPC vesicles, decreasing the accessibility of 12 doxyl with NiAA. To better illustrate these trends Figure 5 1 and Figure 5 2 are plotted as a 1/2 and shown in Figure 5 3 a nd Figure 5 4. Obvious deviations between the 4:1 POPC:POPG and the 4:1 DPPC:POPG data can be seen at the 12 doxyl position in these graphs. Figure 5 1/2 (o xygen) plotted as a function of KL 4 mol e percent. Samples contain either 5 doxyl (left), 7 doxyl (center), or 12 doxyl (right) in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). 5 Doxyl 7 Doxyl 12 Doxyl

PAGE 98

98 Figure 5 1/2 (NiAA) plotted as a funct ion of KL 4 mole percent. Samples contain either 5 doxyl (left), 7 doxyl (center), or 12 doxyl (right) in 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). Effect of KL 4 on Spin label Depth 1/2 is a good way of looking at changes occurring with a specific paramagnetic collider and a spin label; however, looking at the 1/2 for hydrophilic and lipophilic colliders can give a good understanding of the depth of the spin label. One issue when looking at the changes in mobility and accessibility is if the peptide is interacting at that spin label position or if the dynamics of the lipid system have changed, such as a thickening or thinning of the bilayer, which may affect the results. By u ch is discussed extensively in C hapter 2, a depth profile of the doxyl spin label can be graphed as a function of KL 4 1/2 is changing because KL 4 is displacing solvent from around th e spin 1/2 may indicate that the depth of the doxyl spin label is changing, therefore, a thickening or thinnin g of the bilayer may be occurring. Figure 5 4 concentration for both lipid systems and at three doxyl positions. 5 Doxyl 7 Doxyl 12 Doxyl

PAGE 99

99 Figure 5 4 concentration at 5 doxyl (left), 7 do xyl (center), and 12 doxyl (right) positions in both 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). From Figure 5 label increases its depth within the bilayer. For the 5 position on the lip 1/2 (o 1/2 (NiAA) is less than one and the natural log of this will 1/2 be seen moving from 5 doxyl to 12 doxyl. Observing the 5 doxyl graph in Figure 5 5 4 addition in both lipid systems, indicating that the relative depth of the 5 doxyl spin label does not change as KL 4 binds. The 7 doxyl data suggests the same with a slight deviation in the 4:1 POPC:POPG 4 concentrations. This may be due to a slight perturbation in the lipid bilayer but it seems minor as illustrated by Figure 5 6 in und two. Finally at the 12 doxyl position the trend 4 Once again showing that the doxyl positions remain at the same relative depth upon KL 4 binding, therefore, suggesting that the bilayer is neither thickening nor thinning and that the trends seen in mobility and accessibility are due to KL 4 interacting directly with 5 Doxyl 7 Doxyl 12 Doxyl

PAGE 100

100 the spin plotted in Figure 5 with no KL 4 suggesting any perturbation in the depth of the doxyl spin label is minimal. Figure 5 4 concentrat ion at 5 doxyl (left), 7 doxyl (center), and 12 doxyl (right) positions in both 4:1 DPPC:POPG (black triangles) and 4:1 POPC:POPG (grey squares). Insertion Depth of KL 4 The determination of the relative depths of each doxyl spin pre label KL 4 to. This will give a relative depth corresponding to the doxyl spin labels in the two lipid systems, therefore giving a model of KL 4 penetration depth between POPC and DPPC rich ves icles. Power saturation data for all eight KL 4 IAP variants were collected at 2 mol % KL 4 IAP ere calculated as described in C function of spin label position is shown in Figure 5 7. For comparison, spin values are marked with a line for each doxyl position using the same concentration of KL 4 (2 mol percent). 5 Doxyl 7 Doxyl 12 Doxyl

PAGE 101

101 Figure 5 4 IAP plotted as a function of spin label position for 4:1 DPPC:POPG (left) and 4:1 POPC:POPG (right). Spin values are given for the 5 7 and 12 doxyl and shown in green lines. These are used as a ruler to determine the relative depth of KL 4 in each bilayer. As previously stated, the first four spin labels (C7 C10) make a full turn around the 18) and their results should be compared to one anoth er. Figure 5 7 shows that in both DPPC and POPC rich systems, moving from C7 to C9 followed by a decrease at C10 completing the full turn around a helix in the plane of the for DPPC indicate that KL 4 is penetrating deeper in these vesicles than POPC. The next set of spin labeled KL 4 IAP samples are C12 C15 and also make a comp lete turn around the helix. Figure 5 7 shows a similar trend as seen for C7 C10 however much less pronounced. This is expected after analysi s of the mobility results from C hapter 4 which suggests KL 4 may not be lying entirely in the plane of the bilayer but may exhibit a slight tilt causing erratic results at the C12 C15 positions. An important observation is at the C14 position which gave peculiar results in the mobility studies (Figure 4 18). The relative depth as calculated from power saturation show s C14 being deeper in the bilayer when compared to the neighboring amino acid

PAGE 102

102 positions. This correlates well with the proposed helical wheel projections (Figure 4 18) and suggests that the decrease in mobility seen at this position may be due to some hin C omparing the power saturation depth parameter of spin labeled lipids with that of spin labeled peptide allows for the determination of a relative penetration depth between the two lipid s ystems. Figure 5 4 IAP at eight positions in addition to the corresponding spin A model of KL 4 in the two lipid systems is proposed and is shown in Figure 5 8 using the first four spin labeled peptide positions (C7 C10). A slight alteration may need to be made for POPC because of the increase depth calculated at positions C12 C15 when compared with C7 C10 from Figure 5 7. The data suggests KL 4 lying at a slight angle in the POPC rich vesicles with the C12 C15 positions penetrating slightly further into the bilayer when compared to C7 C10. For DPPC vesicles the data in Figure 5 7 suggests KL 4 lies parallel with the bilayer throughout positions C7 C10 and C 12 C15. The peptide model is based on the NMR data previously discussed [42, 86] while using the 7 to determine the side chain penetration depths using the color coded schematic discussed in the Figure 5 8 caption. In 4:1 DPPC:POPG vesicles KL 4 penetrates deeper in the bilayer when compared to 4:1 POPC:POPG allowing for a possible mechanism of DPPC enrichment. In addition, the difference in either lying parallel or being slightly tilted, as suggested from the power saturation data for POPC, may also play a role in KL 4 function by altering its overall binding interaction.

PAGE 103

103 Figure 5 8. Models of KL 4 partitioning into DPPC rich (left) and POPC rich (right) 4 C variant (Figure 5 7). The leucines substituted in this study are color coded with respect to relative partitioning of the I AP spin label at 45 C with L7R1 4 are based on NMR measurements in DPPC rich and POPC rich environments. Conclusions In C hapter 4, the mobility studies suggested what was already seen i n Dr. Long et al NMR studies that KL 4 inserted in the plane of the bilayer and at different depths for DPPC an d POPC rich vesicles. In C hapter 4 power saturation CW EPR was performed to give a better understanding of the relative depths between the two lipid systems. The proposed model in Figure 5 8 is based on the NMR data and the power saturation data collected for both spin labeled lipid and spin labeled KL 4 samples in C hapter 4 It shows that KL 4 inserts deeper in DPPC rich vesicles with the C9 pos ition reaching deep in the bilayer beyond the 12 doxyl position. This difference in binding may play a critical role in what many believe is an enriching of DPPC at the air fluid interface in the alveoli. Understanding these differences of KL 4 and lipid interactions allows for further development of more specific and targeted drug development.

PAGE 104

104 CHAPTER 6 CONCLUSIONS AND FUTU RE DIRECTIONS Conclusions Pulmonary surfactant is a mixture of lipids and proteins that are vital to proper lung function. Defic iencies or mutations in pulmonary surfactant can lead to a wide range of diseases, and one of utmost importance is respiratory distress syndrome (RDS), a condition which commonly occurs in premature infants. According to the center for disease control (CD C), RDS is the fourth leading cause of neonatal mortality and contributes to the longest hospital stay among preterm infants. Current treatment for RDS consists of administering calf lung extract via a breathing tube directly into the lungs of the patient until proper lung function returns. This is of concern because administrating exogenous surfactant to a preterm infant with a compromised immune system can produce undesired effects. Many studies have attempted to remedy this issue by closely studying t he make up of pulmonary surfactant. From these studies it has been shown that the fully saturated DPPC lipid, the negatively charged POPG lipid, and the hydrophobic surfactant protein B (SP B) are of critical importance. Due to the hydrophobicity of SP B it has been difficult to purify, so synthetic constructs mimicking its efficacy are in development. One such mimic is the 21 amino acid peptide KL 4 which is currently in FDA trials. Chapter 1 provides detailed background information relevant to both p ulmonary surfactant and KL 4 The techniques used to examine KL 4 include circular dichroism (CD), continuous wave electron paramagnetic resonance spectroscopy (CW EPR), and power saturation CW EPR. Detailed descriptions on each of these methodologies are given in Chapter 2.

PAGE 105

105 Chapter 3 describes the optimization of t he CW EPR studies described in C hapters 4 and 5. In order to stay consistent with charge in the lipid studies, determination of differences between 3:1 POPC:POPG and 4:1 POPC:POPG needed to be d etermined. In addition, proper temperature and pH studies were performed to allow for increased sensitivity for the power saturation studies. environment upon KL 4 binding, CW EPR mob ility studies were performed using either a doxyl spin label on the lipid acyl chain or employing an IAP spin label to the peptide backbone. Results in C hapter 4 show that KL 4 does not span the bilayer in a transmembrane orientation but lies parallel to t he bilayer normal. In addition, differences are seen between DPPC and POPC rich vesicle systems. As previously indicated by NMR studies performed by Dr. Long et al CW EPR shows KL 4 penetrates deeper into DPPC bilayers interacting at the 12 positi on on the lipid acyl chain. CW EPR mobility results analyzed using multiple semi quantitative parameters each indicate this difference in penetration depth between the two lipid systems. In addition to the CW EPR mobility studies, po wer saturation EPR was use d in C hapter 5 to study spin label solvent accessibility at both the lipid and peptide level and develop a penetration depth profile in the two systems. As with the mobility studies in C hapter 4, power saturation suggests KL 4 penetrates deeper in the DPPC rich bilayers when compared to POPC rich. A penetration model was constructed in both lipid systems as determined by comparing power saturation values of the peptide with that of power saturation values of the lipid acyl chain. In addition, the solvent accessibility trend is indicative of a peptide lying in the plane of the bilayer and not of a peptide in a

PAGE 106

106 transmembrane conformation. A slight tilt deviating from an entirely parallel peptide is suggested in the POPC vesicles, providing another possible difference between these two lipid systems. An important aspect of understanding KL 4 with different lipids. As it has been hypothesized that KL 4 preferentially inserts DPPC at the air fluid interface, understandi ng how it interacts differently with DPPC than other non saturated lipids can allow for a fundamental understanding of KL 4 and possible advancements in future drug development. Future Directions CW EPR Studies on SP B C terminus To date, research has foc used on the terminal ends of SP B, a construct of both N and C terminus called mini B, and a few entirely synthetic mimics like KL 4 Our group has performed CW EPR studies on KL 4 but our collaborator Dr. Long has looked at not only KL 4 but the N and C t ermini of SP B by a variety of NMR techniques. Since EPR and NMR have complemented each other well in studying KL 4 it is inevitable to and C termini. Collaboration has already begun on CW EPR mobility and power satu ration studies, similar to that presented in this dissertation, on the C terminus of SP B. The large discrepancies at the amino acid level between KL 4 and the termini suggest a careful consideration of spin label placement will be needed, especially due t o the presence of prolines and native cysteines. Pulsed EPR on KL 4 by Electron Spin Echo Envelope Modulation (ESEEM) CW EPR power saturation studies allow for an indirect study of the water penetration into the bilayer by use of an aqueous paramagnetic co llider such as NiAA.

PAGE 107

107 To directly look at changes in the water penetration profile a pulsed EPR technique called electron spin echo envelop modulation (ESEEM) can be utilized. ESEEM allows for a direct look at D 2 O bonded to the N O group of the nitroxide spin label. This technique was first established by Dr. Marsh et al and used on a similar system of DPPC but with cholesterol to affect the water penetration profile within the bilayer [160]. This technique does require cryogenic temperatures; therefore it works well in tandem with CW EPR power saturation which can be run at physiological temperatures. CW EPR KL 4 Studies in Different Lipid Systems Surfactant proteins have been studied in a variety of different lipid systems with varying composition. One such composition that has interested our group is a combination of the two systems employed in this di ssertation, DPPC:POPC:POPG (2:2: 1). Studying a lipid system such as this one may show results that resemble one of the two systems presented in this dissertation, giving further insight into KL 4 interaction. Preliminary results of CW EPR mobility and power saturation studies have been collected at the 12 doxyl position and C7 C10 KL 4 IAP.

PAGE 108

108 LIST OF REFERENCES 1. Goerke, J., Pulmonary surfactant: funct ions and molecular composition. Biochimica et Biophysica Acta, 1998(1408): p. 79 89. 2. Piknova, B., V. Schram, and S.B. Hall, Pulmonary surfactant: phase behaivor and function. Current Opinion in Structural Biology, 2002. 12 : p. 487 494. 3. Serrano, A.G. an d J. Perez Gil, Protein lipid interactions and surface activity in the pulmonary surfactant system. Chemistry and Physics of Lipids, 2006(141): p. 105 118. 4. Whitsett, J.A. and T.E. Weaver, Hydrophobic surfactant proteins in lung function and disease. New Englan Journal of Medicine, 2002. 26 (347): p. 2141 2148. 5. Wright, J.R., Immunoregulatory functions of surfactant proteins. Nature Reviews Immunolgy, 2004. 5 : p. 58 68. 6. Iwaarden, F.v., et al., Pulmonary surfactant A enhances the host defense mechanism of rat alveolar macrophages. American Journal of Respiratory Cellular Molecular Biology, 1990. 2 : p. 91 98. 7. Ohmer Schrock, D., et al., Lung surfactant protein A (SP A) activates a phospoinositide/calcium signaling pathway in alveolar macrophages. Journal o f Cell Science, 1995. 108 : p. 3695 3702. 8. Thet, L.A., et al., Changes in the sedimentation of surfactant in ventilated excised rat lungs. Journal of Clinical Investigations, 1979. 64 : p. 600 608. 9. Wright, J.R., Regulation of pulmonary surfactant secretio n and clearance. Annual Review of Physiology, 1991. 53 : p. 395 414. 10. Baughman, R.P., The uncertanties of Bronchoalveolar lavage. European Respiration Journal, 1999. 10 : p. 1940 1942. 11. Holt, P.G., A simple technique for the preparation of high numbers o f viable alveolar macrophages from laboratory animals. Journal of Immunology, 1979. 27 : p. 189 198. 12. Medin, N.I., J.W. Osebold, and Y.C. Zee, A procedure for pulmonary lavage in mice. American Journal of Veterinarians, 1976. 37 : p. 237 238. 13. Reynolds, H.Y. and H.H. Newball, Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage. Journal of Laboratory Clinical Medicine, 1974. 84 : p. 559 573.

PAGE 109

109 14. Rudent, A., et al., Enhancement of bronchoalveolar cell recovery and stimulat ion of alveolar macrophage chemiluminescence and resistance to influenza virus after treatment with RU 41821 aerosol. Antimicrobial Agents and Chemotherapy, 1987. 31 (6): p. 920 924. 15. Nicholas, T.E., J.H. Power, and H.A. Barr, Surfactant homeostasis in th e rat lung during swimming exercise. Journal of Applied Physiology, 1982. 53 : p. 1521 1528. 16. Nicholas, T.E., J.H. Power, and H.A. Barr, The pulmonary consequences of a deep breath. Respiratory Physiology, 1982. 49 : p. 315 324. 17. Clements, J.A. and M.E. Avery, Lung surfactant and neonatal respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine, 1998. 157 : p. S59 S66. 18. Griese, M., Pulmonary surfactant in health and human lung disease: state of the art. European Respirati on Journal, 1999. 13 : p. 1455 1476. 19. Burgess, T.L. and R.B. Kelly, Constitutive and regulated secretion of proteins. Annual Review of Cell Biology, 1987. 3 : p. 243 293. 20. Dobbs, L.G., et al., Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats. Biochimica et Biophysica Acta, 1982. 713 : p. 118 127. 21. Kikkawa, Y., et al., The type II epithelial cells of the lung: chemical composition and phospholipid synthesis. Laboratory Investigation, 1975. 32 : p. 295 302. 22. Mason, R.J., et al., Isolation and propeties of type II alveolar cells from rat lung. American Review of Respiratory Disease, 1977. 115 : p. 1015 1026. 23. Clements, J.A., Functions of the alveolar lining. American Review of Respiratory Disease, 1977. 115 : p. 67 71 24. Johansson, J. and T. Curstedt, Molecular structures and interactions of pulmonary surfactant components. European Journal of Biochemistry, 1997. 3 (244): p. 675 693. 25. Johansson, J., et al., The NMR structure of pulmonary surfactant associated polypep tide SP C in an apolar solvent contain a valyl Biochemistry, 1994. 33 : p. 6015 6023. 26. Postle, A.D., E.L. Heeley, and D.C. Wilton, A comparison of the molecular species compositions of mammalian lung surfactant phospholipids. Comparative Bio chemistry and Physiology, 2001. 129 : p. 65 73. 27. Wright, J.R. and J.A. Clements, Metabolism and turnover of lung surfactant. American Review of Respiratory Disease, 1987. 135 : p. 426 444.

PAGE 110

110 28. Ikegami, M., Surfactant catabolism. Respirology, 2006. 11 : p. S2 4 S27. 29. Gunther, A., et al., Surfactant alteration and replacement in acute respiratory distress syndrome. Respiratory Research, 2001. 2 (6): p. 353 364. 30. Wright, J.R., Pulmonary surfactant: a front line of lung host defense. Journal of Clinical Investi gations, 2003. 111 : p. 1453 1455. 31. Fujiwara, T., et al., Artificial surfactant therapy in hyaline membrane disease. Lancet, 1980: p. 55 59. 32. Halliday, H.L., History of surfactant from 1980. Biology of the Neonate, 2005. 87 : p. 317 322. 33. Soll, R.F., P rophylactic natural surfactant extract for preventing morbidity and mortality in preterm infants. Cochrane Database System Reviews, 1997. 4 34. Soll, R.F., Multiple versus single dose natural surfactant extract for severe neonatal respiratory distress synd rom. Cochrane Database System Reviews, 1999. 2 35. Soll, R.F. and F. Blanco, Natural surfactant extract versus synthetic surfactant for neonatal respiratory distress syndrome. Cochrane Database System Reviews, 2001. 2 36. Soll, R.F. and P. Dargaville, Surf actant for meconium aspiration syndrome in full term infants. Cochrane Database System Reviews, 2000. 2 37. Soll, R.F. and C.J. Morley, Prophylactic versus selective use of surfactant in preventing morbidity and mortality in preterm infants. Cochrane Datab ase System Reviews, 2001. 2 38. Stevens, T.P., M. Blennow, and R.F. Soll, Early surfactant treatment with brief ventilation versus selective surfactant and continued mechanical ventilation for preterm infants with or at risk of RDS. Cochrane Database Syste m Reviews, 2004. 3 39. Yost, C.C., Early versus delayed selective surfactant treatment for neonatal respiratory distress syndrome. Cochrane Database System Reviews, 1999. 4 40. Zhu, Y., et al., KL4 surfactant (Lucinactant) protects human airway epithelium from hyperoxia. Pediatric Reseach, 2008. 64 (2): p. 154 158. 41. Gunning, P.A., et al., Effect of surfactant type on surfactant protein interactions at the air water interface. Biomacromolecules, 2004. 5 : p. 984 991. 42. Mills, F.D., et al., The helical struc ture of surfactant peptide KL4 when bound to POPC:POPG lipid vesicles. Biochemistry, 2008. 47 : p. 8292 8300.

PAGE 111

111 43. Artigas, A., et al., The American European consensus conference on ARDS, part 2. American Journal of Respiratory and Critical Care Medicine, 199 8. 157 : p. 1332 1347. 44. Hudson, L.D. and K.P. Steinberg, Epidemiology of acute lung injury and ARDS. Chest, 1999. 116 : p. 74S 82S. 45. Lewis, J.F. and R. Veldhuizen, The role of exogenous surfactant in the treatment of acute lung injury. Annual Review of P hysiology, 2003. 65 : p. 613 642. 46. Suchyta, M.R., et al., The adult resporatory distress syndrome. A report of survival and modifying factors. Chest, 1992. 101 : p. 1074 1079. 47. Halliday, H.L., Recent clinical trials of surfactant treatment for neonates. Biology of the Neonate, 2006. 89 : p. 323 329. 48. Obladen, M., History of surfactant up to 1980. Biology of the Neonate, 2005. 87 : p. 308 316. 49. Revak, S.D., et al., Use of human surfactant low molecular weight apoproteins in the reconstitution of surfact ant biologic activity. Journal of Clinical Investigations, 1988. 81 : p. 826 833. 50. Robertson, B. and H.L. Halliday, Principles of surfactant replacement. Biochimica et Biophysica Acta, 1998. 1408 : p. 346 361. 51. Centers for Disease Control and Prevention Web site 2010; Available from: http://www.cdc.gov/nchs 52. Klaus, M.H., J.A. Clements, and R.J. Havel, Composition of surface active material isolated from beef lung. Proceedings of the National Academy of Sciences 1961. 47 : p. 1858 1859. 53. Goerke, J. and J.A. Clements, Alveolar surface tension and lung surfactant. Handbook of Comprehensive Physiology, 1986: p. 247 261. 54. Malcolm, J.D. and C.D. Elliott, Interfacial tension from height and diameter of a single ses sile drop or captive bubble. The Canadian Jornal of Chemical Engineering, 1980. 58 (2): p. 151 153. 55. Schurch, S., et al., Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hysteresis, stability. Biochimica et Biophysica Acta, 1992. 1103 : p. 127 136. 56. Engle, M.J., R.L. Sanders, and W.J. Longmore, Phospholipid composition and acyltransferase activity of lamellar bodies isolated from rat lung. Biochemistry and Biophysics, 1976. 173 : p. 586 595.

PAGE 112

112 57. Hallman, M. and L. Gluck, Phosphatidylglycerol in lung surfactant. Possible modifier of surfactant function. The Journal of Lipid Research, 1976. 17 : p. 257 262. 58. Rooney, S.A., P.M. Canavan, and E.K. Motoyama, The identification of phosphatidylglycerol in the rat, rabbit, monkey, and human lung. Biochimica et Biophysica Acta, 1974. 360 : p. 56 67. 59. Hallman, M., B. Feldman, and L. Gluck, The absence of phospatidylglycerol in surfactant. Pediatric Reseach, 1975. 9 : p. 396. 60. Brogden, K.A., et al., Isolation of an ov ine pulmonary surfactant associated anionic peptide bactericidal for Pasteurella haemolytica. Proceedings of the National Academy of Sciences, 1996. 93 : p. 412 416. 61. Iwaarden, J.F.V., et al., Rat surfactant protein D enhances the production of oxygen rad icals by rat alveolar macrophages. Biochemical Journal, 1992. 286 : p. 5 8. 62. Manz Keinke, H., H. Plattner, and J. Schlepper Schafer, Lung surfactant protein A (SP A) enhances serum independent phagocytosis of bacteria by alveolar macrophages. European Jou rnal of Cell Biology, 1992. 57 (1): p. 95 100. 63. Tenner, A.J., et al., Human pulmonary surfactant protein (SP A), a protein structurally homologous to C1q, can enhance FcR and CR1 mediated phagocytosis. The Journal of Biological Chemistry, 1989. 264 (23): p. 13923 13928. 64. Zimmerman, P.E., et al., 120 kD Surface glycoprotein of Pneumocystis carinii is a ligand for surfactant protein A. Journal of Clinical Investigations, 1992. 89 : p. 143 149. 65. Oosterlaken Dijksterhuis, M.A., et al., Characterization of l ipid insertion into monomolecular layers mediated by lung surfactant proteins SP B and SP C. Biochemistry, 1991. 30 : p. 10965 10971. 66. Poulain, F.R., S. Nir, and S. Hawgood, Kinetics of phospholid membrane fusion induced by surfactant apoproteins A and B. Biochimica et Biophysica Acta, 1996. 1278 : p. 169 175. 67. Taneva, S.G. and K.M.W. Keough, Dynamic surface properties of pulmonary surfactant proteins SP B and SP C and their mixtures with dipalmitoylphosphatidylcholine. Biochemistry, 1994. 33 : p. 14660 14 670. 68. Wang, Z., et al., Differential activity and lack of synergy of lung surfactant proteins SP B and SP C in interactions with phospholipids. The Journal of Lipid Research, 1996. 37 : p. 1749 1760. 69. Yukitake, K., et al., Surfactant apoprotein A modifi es the inhibitory effect of plasma proteins on surfactant activity in vivo. Pediatric Reseach, 1995. 37 (1): p. 21 25.

PAGE 113

113 70. Wong, C.J., et al., Localization and developmental expression of surfactant proteins D and A in the respiratory tract of the mouse. Ped iatric Reseach, 1996. 39 (6): p. 930 937. 71. Hatzis, D., et al., Human surfactant protein C: Genetic homogeneity and expression in RDS comparison with other species. Experimental Lung Research, 1994. 20 (1): p. 57 72. 72. Akinbi, H.T., et al., Rescue of SP B knockout mice with a truncated SP B proprotein. The Journal of Biological Chemistry, 1997. 272 (15): p. 9640 9647. 73. Clark, J.C., et al., Decreased lung compliance and air trapping in heterozygous SP B deficient mice. American Journal of Respiratory Cell a nd Molecular Biology, 1997. 16 (1): p. 46 52. 74. Grossman, G., et al., Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B. Journal of Applied Physiology, 1997. 82 : p. 2003 2010. 75. Tokieda, K., et al., Pulmonary d ysfunction in neonatal SP B deficient mice. American Journal of Physiological Lung and Cell Molecular Physiology, 1997. 273 : p. 875 882. 76. Weaver, T.E. and J.J. Conkright, Functions of Surfactant Proteins B and C. Annual Review of Physiology, 2001. 63 : p. 555 578. 77. Kuroki, Y. and H. Sano, Functional roles and structural analysis of lung collectins SP A and SP D. Biology of the Neonate, 1999. 76 : p. 19 21. 78. Wetering, J.K.v.d., L.M.G.v. Golde, and J.J. Batenburg, Collectins: Players of the innate immune system. European Journal of Biochemistry, 2004. 271 : p. 1229 1249. 79. Korfhagen, T.R., et al., Altered surfactant function and structure in SP A gene targeted mice. Proceedings of the National Academy of Sciences, 1996. 93 : p. 9594 9599. 80. Korfhagen, T.R. A.M. LeVine, and J.A. Whitsett, Surfactant protein A (SP A) gene targeted mice. Biochimica et Biophysica Acta, 1998. 1408 (2 3): p. 296 302. 81. Korfhagen, T.R., et al., Surfactant protein D regulates surfactant phospholipid homeostasis in vivo. Journal of Biological Chemistry, 1998. 273 : p. 28438 28443. 82. McCormack, F.X., et al., Alanine mutagenesis of surfactant protein A reveals that lipid binding and pH dependent liposome aggregation are mediated by the carbohydrate recognition domain. Biochemistry, 19 97. 36 : p. 13963 13971.

PAGE 114

114 83. Robertson, B., J. Johansson, and T. Curstedt, Synthetic surfactants to treat neonatal lung diesease. Molecular Medicine Today, 2000. 6 : p. 1 6. 84. Seurynck, S.L., J.A. Patch, and A.E. Barron, Simple, helical peptoid analogs of lu ng surfactant protein B. Chicstry & Biology, 2005. 12 : p. 77 88. 85. Antharam, V.C., et al., Penetration depth of surfactant peptide KL4 into membranes is determined by fatty acid saturation. Biophysical Journal, 2009. 96 : p. 4085 4098 86. Long, J.R., et al. Partitioning, dynamics, and orientation of lung surfactant peptide KL 4 in phospholipid bilayers. Biochimica et Biophysica Acta, 2010. 1798 : p. 216 222. 87. Lipp, M.M., et al., Phase and morphology changes in lipid monolayers induced by SP B protein and it s amino terminal peptide. Science, 1996. 273 : p. 1196 1199. 88. Baatz, J.E., et al., Effects of surfactant associated protein SP B synthetic analogs on the structure and surface activity of model membrane bilayers. Chemistry and Physics of Lipids, 1991. 60 : p. 163 178. 89. Bruni, R., H.W. Taeusch, and A.J. Waring, Sufactant protein B: lipid interactions of synthetic peptides representing the amino terminal amphipathic domain. Proceedings of the National Academy of Sciences, 1991. 88 : p. 7451 7455. 90. Lipp, M. M., et al., Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers. Biophysical Journal, 1991. 72 : p. 2783 2804. 91. Longo, M.L., et al., A function of lung surfactant protein SP B. Scie nce, 1993. 261 : p. 453 456. 92. Revak, S.D., et al., The use of synthetic peptides in the formation of biophysically and biologically active pulmonary surfactants. Pediatric Reseach, 1991. 29 : p. 460 465. 93. Waring, A., et al., Synthetic amphipathic sequenc es of surfactant protein B mimic several physiochemical and in vivo properties of native pulmonary surfactant proteins. Peptide Research, 1989. 2 : p. 308 313. 94. Cochrane, C.G. and S.D. Revak, Pulmonary surfactant protein B (SP B): structure function relat ionships. Science, 1991. 254 : p. 566 568. 95. Gustafsson, M., et al., The 21 residue surfactant peptide (LysLeu 4 ) 4 Lys(KL 4 ) is a helix with a mixed nonpolar/polar surface. Federation of European Biochemical Societies, 1996. 384 : p. 185 188. 9 6. Cochrane, C.G., et al., Bronchoalveolar lavage with KL4 surfactant in models of meconium aspiration syndrome. Pediatric Reseach, 1998. 44 : p. 705 715.

PAGE 115

115 97. Revak, S.D., et al., Efficacy of KL4 surfactant in premature infant monkeys. Pediatric Reseach, 1995 37 : p. A347 A347. 98. Revak, S.D., et al., Efficacy of synthetic peptide containing surfactant in the treatment of respiratory distress syndrome in preterm infant rhesus monkeys. Pediatric Reseach, 1996. 39 (715 724). 99. Walther, F.J., et al., Spiking Surv anta with synthetic surfactant peptides improves oxygenation in surfactant deficient rats. American Journal of Respiratory and Critical Care Medicine, 1997. 156 : p. 855 861. 100. Walther, F.J., et al., Protein composition of synthetic surfactant affects gas exchange in surfactant deficient rats. Pediatric Reseach, 1998. 43 : p. 666 673. 101. Cochrane, C., et al., The efficacy and safety of KL4 surfactant in preterm infants with respiratory distress syndrome. Respiratory Critical Care Medicine, 1996. 153 : p. 40 4 410. 102. Mansour, H.M., S. Damodaran, and G. Zografi, Characterization of the in situ structural and interfacial properties of the cationic hydrophobic heteropolypeptide, KL4, in lung surfactant bilayer and monolayer models at the air water interface: Im plications for pulmonary surfactant delivery. Molecular Phamaceutics, 2008. 5 (5): p. 681 695. 103. Wiswell, T.E., et al., Bronchopulmonary segmental lavage with surfaxin (KL4 surfactant) for acute respiratory distress syndrome. American Journal of Respirato ry and Critical Care Medicine, 1999. 160 : p. 1188 1195. 104. Moya, F.R., et al., A multicenter, randomized, masked, comparison trial of lucinactant, colfosceril palmitate, and beractant for the prevention of respiratory distress syndrome among very preterm infants. Pediatrics, 2005. 115 : p. 1018 1029. 105. Saenz, A., et al., Physical properties and surface activity of surfactant like membranes containing the cationic and hydrophobic peptide KL 4 FEBS J., 2006. 273 : p. 2515 2527. 106. Vogt, B., et al., The topo logy of lysine containing amphipathic peptides in bilayers by circular dichroism, solid state NMR, and molecular modeling. Biophysical Journal, 2000. 79 : p. 2644 2656. 107. Martinez Gil, L., J. Perez Gil, and I. Mingarro, The surfactant peptide KL 4 sequence is inserted with a transmembrane orientation into the endoplasmic reticulum membrane. Biophysical Journal, 2008. 95 : p. L 36 L38. 108. Hessa, T., et al., Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature, 2005. 433 : p. 37 7 381.

PAGE 116

116 109. Hessa, T., et al., Molecular code for transmembrane helix recognition by the Sec61 translocon. Nature, 2007. 450 : p. 1026 1030. 110. Cai, P., C.R. Flach, and R. Mendelsohm, An infrared reflection absorption spectroscopy study of the secondary str ucture in (KL 4 ) 4 K, a therapeutic agent for respiratory distress syndrom, in aqueous monolayers with phospholipids. Biochemistry, 2003. 42 : p. 9446 9452. 111. Houghten, R.A., General method for the rapid solid phase synthesis of large numbers of peptides: sp ecificity of antigen antibody interaction at the level of individual amino acids. 1985, 1985. 82 : p. 5131 5135. 112. Merrifield, R.B., Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of American Chemical Society, 1963. 85 : p. 2149 2154. 113. Edman, P., Method for determination of the amino acid sequence in peptides. Acta Chemica Scandinavica, 1950. 4 : p. 283 293. 114. Niall, H.D., Automated degradation: The protein sequenator. Methology of Enzymology, 1973. 27 : p. 942 1010. 115. McCon nell, H.M., et al., Spin Labeled Biomolecules. PNAS, 1965. 54 (4): p. 1010 1017. 116. Altenbach, C., et al., A collision gradient method to determine the immersion depth of nitroxides in lipid bilayers: Application to spin labeled mutants of bacteriorhodopsi n. Proceedings of the National Academy of Sciences, 1994. 91 : p. 1667 1671. 117. Hubbell, W.L. and C. Altenbach, Investigation of structure and dynamics in membrane proteins using site directed spin labeling. Current Opinion in Structural Biology, 1994. 4 (4 ): p. 566 573. 118. Hubbell, W.L., D.S. Cafiso, and C. Altenbach, Identifying conformational changes with site directed spin labeling. Nature Structural Biology, 2000. 7 (9): p. 735 739. 119. Hubbell, W.L., et al., Recent advances in site directed spin labeli ng of proteins. Current Opinion in Structural Biology, 1998. 8 : p. 649 656. 120. Hubbell, W.L. and H.M. McConnell, Molecular Motion in Spin Labeled Phospholipids and Membranes. Journal of the American Chemical Society, 1971. 93 (2): p. 314 326. 121. Klug, C.S ., et al., Ligand induced conformational change in the ferric enterobactin receptor FepA as studied by site directed spin labeling and time domain ESR. Biochemistry, 1998. 37 : p. 9016 9023.

PAGE 117

117 122. Langen, R., et al., Crystal structures of spin labeled T4 lyso zyme mutants: Implications for the interpretation of EPR spectra in terms of structure. Biochemistry, 2000. 39 : p. 8396 8405. 123. Voss, J., et al., A method for distance determination in proteins using a designed metal ion binding site and site directed sp in labeling: Evaluation with T4 lysozyme. Proceedings of the National Academy of Sciences, 1995. 92 : p. 12295 12299. 124. Hubbell, W.L., et al., Watching proteins move using site directed spin labeling. Structure, 1996. 4 : p. 779 780. 125. Czogalla, A., et a l., Attaching a spin to a protein site directed spin labeling in structural biology. Acta Biochimica Polonica, 2007. 54 (2): p. 235 244. 126. Fanucci, G.E. and D.S. Cafiso, Recent advances and applications of site directed spin labeling. Current Opinion in Structural Biology, 2006. 16 : p. 644 653. 127. Mchaourab, H.S., et al., Motion of spin labeled side chains in T4 lysozyme: Effect of side chain structure. Biochemistry, 1999. 38 : p. 2947 2955. 128. Jost, P., et al., Lipid spin labels in lecithin multilayers A study of motion along fatty acid chains. Journal of Molecular Biology, 1971. 59 : p. 77 98. 129. Keana, J.F.W., Newer aspects of the synthesis and chemistry of nitroxide spin labels. Chemical Reviews, 1978. 78 (1): p. 37 64. 130. Libertini, L.J., et al., Orientation of lipid spin labels in lecithin multilayers. PNAS, 1969. 64 (1): p. 12 19. 131. Marsh, D. and A. Watts, Lipid protein interactions. Wiley Interscience, 1982. 2 132. Snel, M.M.E. and D. Marsh, Accessibility of spin labeled phospholipids in anioni c and zwitterionic bilayer membranes to paramagnetic relaxation agents. Continuous wave power saturation EPR studies. Biochimica et Biophysica Acta, 1993. 1150 : p. 155 161. 133. Atkins, P. and J.d. Paula, Elements of Physical Chemistry, 4th Ed. 2005: Oxfor d University Press. 134. Fasman, G.D., Circular dichroism and the conformational analysis of biomolecules 1996: Springer. 135. Applied Photophysics 2003. 136. Greenfield, N. and G.D. Fasman, Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry, 1969. 8 : p. 4108 4116.

PAGE 118

118 137. Sreerama, N. and R.W. Woody, A self consistent method for the analysis of protein secondary structure from circular dichroism. Analytical Biochemistry, 1993. 209 : p. 32 44. 138. Gillard, R.D., Circular dichroism. Analyst, 1963. 88 : p. 825 828. 139. Jongh, H.H.J.d., E. Goormaghtigh, and J.A. Killian, Analysis of circular dichroism spectra of oriented protein lipid complexes: Toward a general application. Biochemistry, 1994. 33 : p. 14521 14528. 140. Lenard, J. and S.J. Singer, Protein conformation in cell membrane preparations as studied by optical rotatory dispersion and circular dichroism. Biochemistry, 1966. 56 : p. 1828 1835. 141. Bruker Biospin 2011. 142. Columbus, L. and W.L. Hubbell, A new spin on prot ein dynamics. Trends in Biochemical Sciences, 2002. 27 (6): p. 288 295. 143. Mchaourab, H.S., et al., Motion of spin labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry, 1996. 35 : p. 7692 7704. 144. Schorn, K. an d D. Marsh, Extracting order parameters from powder EPR lineshapes for spin labelled lipids in membranes. Spectochimica Acta, 1997. 53 : p. 2235 2240. 145. Brown, M.F., J. Seelig, and U. Haberlen, Structural dynamics in phospholipid bilayers from deuterium s pin lattice relaxation time measurements. Journal of Chemical Physics, 1979. 70 (11): p. 5045 5053. 146. Griffith, O.H., P.J. Dehlinger, and S.P. Van, Shape of the hydrophobic barrier of phospholipid bilayers (Evidence of water penetration in biological memb ranes). Journal of Membrane Biology, 1974. 15 : p. 159 192. 147. Hyde, J.S., C.A. Popp, and S. Schreier, Frontier of Biological Engineering, 1978. 2 : p. 1253 1261. 148. Altenbach, C., et al., A collision gradient method to determine the immersion depth of nit roxides in lipid bilayers: Application to spin labeled mutants of bacteriorhodopsin. Biophysics, 1994. 91 : p. 1667 1671. 149. Horie, T. and J. Hildebrandt, Dynamic compliance, limit cycles, and static equilibria of excised cat lung. Journal of Applied Physi ology, 1971. 31 : p. 423 430. 150. Ramamoorthy, A., et al., Solid state NMR investigation of the membrane disrupting mechanism of antimicrobial peptides MSI 78 and MSI 594 from magainin and melittin. Biophysical Journal, 2006. 91 : p. 206 216.

PAGE 119

119 151. Terzi, E., G. Holzemann, and J. Seelig, amyloid peptide (1 40) with lipid membranes. Biochemistry, 1997. 36 : p. 14845 14852. 152. Wieprecht, T., et al., helix coil transition of amphipathic peptides in a membrane envi ronment: implications for the peptide membrane binding equilibrium. Journal of Molecular Biology, 1999. 294 (3): p. 785 794. 153. Veldhuizen, R., et al., The role of lipids in pulmonary surfactant. Biochimica et Biophysica Acta, 1998. 1408 : p. 90 108. 154. Ba atz, J.E., B. Elledge, and J.A. Whitsett, Surfactant protein SP B induces ordering at the surface of model membrane bilayers. Bichemistry, 1990. 29 : p. 6714 6720. 155. Egberts, J., H. Sloot, and A. Mazure, Minimal surface tension, squeeze out and transition temperatures of binary mixtures of dipalmitoylphosphatidylcholine and unsaturated phospholipids. Biochimica et Biophysica Acta, 1989. 1002 : p. 109 113. 156. Meban, C., Effect of lipids and other substances on the adsorption of dipalmitoyl phosphatidyl chol ine. Pediatric Reseach, 1981. 15 : p. 1029 1031. 157. Possmayer, F., Physichemical aspects of pulmonary surfactant. Fetal and Neonatal Physiology, 1997. 115 : p. 1259 1275. 158. Qanbar, R., et al., Role of the palmitoylation of surfactant associated protein C in surfactant film formation and stability. American Journal of Physiology, 1996. 271 : p. L572 L580. 159. Yu, S.H. and F. Possmayer, Effect of pulmonary surfactant protein B (SP B) and calcium on phospholipid adsorption and squeeze out of phosphatidylglycer ol from binary phospholipid monolayers containing dipalmitoylphosphatidylcholine. Biochimica et Biophysica Acta, 1992. 1126 : p. 26 34. 160. Erilov, D.A., et al., Water concentration profiles in membranes measured by ESEEM of spin labeled lipids. Journal of Physical Chemistry, 2005. 109 : p. 12003 12013.

PAGE 120

120 BIOGRAPHICAL SKETCH Austin Lisle Turner was born in Evanston, Illinois. The middle child of three males, he grew up in the Northwest suburbs of Chicago, Illinois, graduating from Adlai E. Stevenson High Sch ool i n 1999. He earned his B.S. in c hemistry from the University of Wisconsin at Whitewater in 2005 after transferring after two year at Bradley University. Up on graduating with his B.S. in c hemistry he entered the pharmaceutical field at Abbott Laborator ies in North Chicago. After beginning as an intern he worked his way up i n the Department of Drug Metabolism as a research associate. After acquiring a broad area of knowledge in the fields of biochemistry and analytical chemistry he opted to pursue a do ctoral degree at the University of Florida. Upon completion of his Ph.D. program, Austin will be looking to re enter th e pharmaceutical industry as a research a ssociate.