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dc.contributor.advisorPace, C. Nicken_US
dc.creatorTrefethen, Jared M.en_US
dc.date.accessioned2005-02-17T21:01:19Z
dc.date.available2005-02-17T21:01:19Z
dc.date.created2004-12en_US
dc.date.issued2005-02-17
dc.identifier.urihttp://hdl.handle.net/1969.1/1431
dc.description.abstractThe primary objective was to study the kinetics of folding of RNase Sa. Wild-type RNase Sa does not contain tryptophan. A tryptophan was substituted at residue 81 (WT*) to allow fluorescence spectroscopy to be used to monitor folding. This tryptophan mutation did not change the stability. An analysis of the folding kinetics of RNase Sa showed two folding phases, indicating the presence of an intermediate and consistent with the following mechanism: D ↔ I ↔ N. Both refolding limbs of the chevron plot (abcissa = final conc. of denaturant and ordinate = kinetic rate) had non-zero slopes suggesting that proline isomerization was not rate-limiting. The conformational stability of a charge-reversed variant, WT*(D17R), of a surface exposed residue on RNase Sa has been studied by equilibrium techniques. This mutant with a single amino acid charge reversal of a surface exposed residue resulted in decreased stability. Calculations using Coulomb’s Law suggested that favorable electrostatic interactions in the denatured state were the cause for the decreased stability for the charge-reversed variant. Folding and unfolding kinetic studies were designed and conducted to study the charge-reversal effect. Unfolding kinetics showed a 10-fold increase in the unfolding rate constant for WT*(D17R) over WT* and no difference in the rate of refolding. Kinetics experiments were also conducted at pH 3 where protonation of Asp17 (charge reversal site) would be expected to negate the observed kinetic effect. At pH 3 the kinetics of unfolding of WT* RNase Sa and the WT*(D17R) mutant were more similar. These kinetic results indicate that a single-site charge reversal lowered the free energy of the denatured state as suspected. Additionally, the results showed that the transition state was stabilized as well. These results show that a specific Coulombic interaction lowered the free energy in the denatured and transition state of the charge-reversal mutant, more than in WT*. To our knowledge, this is the first demonstration that a favorable electrostatic interaction in the denatured state ensemble has been shown to influence the unfolding kinetics of a protein.en_US
dc.format.extent915919 bytes
dc.format.mediumelectronicen_US
dc.format.mimetypeapplication/pdf
dc.language.isoen_USen_US
dc.publisherTexas A&M Universityen_US
dc.subjectprotein foldingen_US
dc.subjectprotein folding kineticsen_US
dc.subjectprotein stabilityen_US
dc.subjectcharge-reversalen_US
dc.subjectdenatured stateen_US
dc.titleThe folding kinetics of ribonuclease Sa and a charge-reversal varianten_US
dc.typeBooken
dc.typeThesisen
thesis.degree.departmentBiochemistry and Biophysicsen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorTexas A&M Universityen_US
thesis.degree.nameMaster of Scienceen_US
thesis.degree.levelMastersen_US
dc.contributor.committeeMemberRussell, David H.en_US
dc.contributor.committeeMemberScholtz, J. Martinen_US
dc.type.genreElectronic Thesisen_US
dc.type.materialtexten_US
dc.format.digitalOriginborn digitalen_US


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