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Surface charges contribution to protein stability of Thermococcus celer L30e. / CUHK electronic theses & dissertations collection

Electrostatic interaction has long been proposed to be an important factor for stabilizing protein. Charge-charge interaction may especially be important to the thermostability of protein, as having more surface electrostatic interactions is one of the common structural features found in thermophilic proteins when compared to their mesophilic homologues. In order to quantitatively investigate the electrostatic contribution to protein stability, two complementary approaches, namely the double mutant cycle approach and pKa shift approach, were carried out. / In the double mutant cycle approach, the coupling free energies of two salt bridges (E6/R92 and K46/E62) and one a long range ion pair (E90/R92) were estimated by using circular dichroism, to find out the thermodynamic parameters of the protein model Thermococcus celer L30e and its charge-to-neutral mutants. It was found that the coupling free energy was temperature independent and was about 3 kJ mol-1 per salt bridge. By using a novel analysis of double mutant cycle of DeltaC p, it was also found that the interaction of salt bridge plays an important role in the reduction of DeltaCp. The temperature independency of coupling free energy and the effect of reducing DeltaCp could explain the general observation very well that thermophilic proteins have highly up-shifted protein stability curves is due to its elevated electrostatic interactions when compared with their mesophilic homologs. / In the pKa shift approach, the native state pKa values of acidic residues were obtained by fitting the side chain carboxyl 13C chemical shifts to microscopic model or global fitting of titrational event (GloFTE), whereas the denatured state pKa values were obtained by conventional pH titration of terminal protected 5-residue glycine-based model peptide. It was found that the surface charge-charge interactions, either attractive or repulsive, were strong and complicated because of the high surface charge density of T. celer L30e. However, the fact that most of the acidic residues have significantly downshifted native state pK a values indicated the surface charge distribution of T. celer L30e is optimized for stabilizing the protein. In addition, we have shown that temperature has negligible effect on pKa values in both native state and denatured state, therefore temperature can only marginally amplify the stabilizing effect in linear manner. / To overcome the unwanted crystallization problem of wild-type T. celer L30e in the low ionic strength neutral pH NMR conditions, which were essential for the pKa shift approach, a quintuple Arg-to-Lys variant was designed to dramatically improve the crystalline solubility, while the surface charges, as well as the structural, thermodynamic, and electrostatic properties, were conserved. It has also shown that electrostatic interaction played a critical role in crystallization at low ionic strength conditions, and arginine residue was especially important in crystal packing because of its high ability of forming salt bridges and hydrogen bonds. / Wild-type T. celer L30e has also shown to have no observable residual structure in the guanidine HC1-induced denatured state, indicating that denatured state of T. celer L30e should not have large effect on the overall protein stability. / Chan, Chi Ho. / Adviser: Kam Bo Wong. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 202-218). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344625
Date January 2010
ContributorsChan, Chi Ho, Chinese University of Hong Kong Graduate School. Division of Life Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, theses
Formatelectronic resource, microform, microfiche, 1 online resource (xv, 218 leaves : ill.)
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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