Escherichia coli CspA is a member of the
cold shock protein family. All cold shock proteins studied
to date fold rapidly by an apparent two-state mechanism.
CspA contains an unusual cluster of aromatic amino acids
on its surface that is necessary for nucleic acid binding
and also provides stability to CspA (Hillier et al., 1998).
To elucidate the role this aromatic cluster plays in the
determining the folding rate and pathway of CspA, we have
studied the folding kinetics of mutants containing either
leucine or serine substituted for Phe18, Phe20, and/or
Phe31. The leucine substitutions are found to accelerate
folding and the serine substitutions to decelerate folding.
Because these residues exert effects on the free energy
of the folding transition state, they may be necessary
for nucleating folding. They are not responsible, however,
for the very compact, native-like transition state ensemble
seen in the cold shock proteins, as the refolding rates
of the mutants all show a similar, weak dependence of unfolding
rate on denaturant concentration. Using mutant cycle analysis,
we show that there is energetic coupling among the three
residues between the unfolded and transition states, suggesting
that the cooperative nature of these interactions helps
to determine the unfolding rate. Overall, our results suggest
that separate evolutionary pressures can act simultaneously
on the same group of residues to maintain function, stability,
and folding rate.