The folding kinetics of ribonuclease Sa and a charge-reversal variant

Show full item record

Title: The folding kinetics of ribonuclease Sa and a charge-reversal variant
Author: Trefethen, Jared M.
Abstract: The 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 .
URI: http : / /hdl .handle .net /1969 .1 /1431
Date: 2005-02-17


The folding kinetics of ribonuclease Sa and a charge-reversal variant. Available electronically from http : / /hdl .handle .net /1969 .1 /1431 .

Files in this item

Files Size Format View
etd-tamu-2004C-BICH-Trefeth.pdf 915.9Kb application/pdf View/Open

This item appears in the following Collection(s)

Show full item record

Search DSpace

Advanced Search