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

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Material Information

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

Thesis/Dissertation Information

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

Subjects

Subjects / Keywords:
crystallography -- dsc -- kinetics -- protein -- thermalstability
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre:
Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

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

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2014
System ID:
UFE0046681:00001