Structural analysis of yeast HSF by site-specific crosslinking

We have introduced cysteine substitutions into the yeast HSF1 gene at a var iety of locations. Most have no phenotypic effect, and therefore provide si te-specific probes for thiol-specific reagents. Crosslinking of single muta nts identifies locations where equivalent regions of individual monomers ca n approach each other in the HSF trimer. Crosslinking of double mutants ind icates regions that can approach closely within a single subunit. Results f or the DNA binding domain and trimerization domain are consistent with know n structural information, and provide essential controls on the validity of the technique. In contrast to these two domains, the N-terminal and C-term inal domains, wherein Lie the transcriptional activators, are highly flexib le, and do not appear to be in stable contact with any other portions of th e protein. None of these patterns are affected by the conformational change that is induced by superoxide or heat shock. We suggest a new model for th e mechanism of HSF regulation that accomodates the structural information p rovided by these studies. (C) 2000 Academic Press.