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 Coulombs
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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/1431 |
| Date | 17 February 2005 |
| Creators | Trefethen, Jared M. |
| Contributors | Pace, C. Nick |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 915919 bytes, electronic, application/pdf, born digital |
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