The DNA-binding domain of the yeast heat shock
transcription factor (HSF) contains a strictly conserved
proline that is at the center of a kink. To define the
role of this conserved proline-centered kink, we replaced
the proline with a number of other residues. These substitutions
did not diminish the ability of the full-length protein
to support growth of yeast or to activate transcription,
suggesting that the proline at the center of the kink is
not conserved for function. The stability of the isolated
mutant DNA-binding domains was unaltered from the wild-type,
so the proline is not conserved to maintain the stability
of the protein. The crystal structures of two of the mutant
DNA-binding domains revealed that the helices in the mutant
proteins were still kinked after substitution of the proline,
suggesting that the proline does not cause the α-helical
kink. So why are prolines conserved in this and the majority
of other kinked α-helices if not for structure, function,
or stability? The mutant DNA-binding domains are less soluble
than wild-type when overexpressed. In addition, the folding
kinetics, as measured by stopped-flow fluorescence, is
faster for the mutant proteins. These two results support
the premise that the presence of the proline is critical
for the folding pathway of HSF's DNA-binding domain.
The finding may also be more general and explain why kinked
helices maintain their prolines.