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Description:
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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 . |