Assessment of solvent exposure of native cysteines in human Hsp90 using thiol-reactive functional tags

Abstract

The biological functions of the molecular chaperone Hsp90 are driven by its complex conformational dynamics. However, the structural features of these dynamics under physiological, solution-phase conditions remain poorly characterized. Here, we present a systematic profiling of native cysteine solvent accessibility in wildtype Hsp90β using a varity of thiol-reactive chemical probes. The results identified Cys366 as the most reactive and solvent-exposed native cysteine. Notably, the accessibility of this residue is unaffected by nucleotide binding or hydrolysis, suggesting its local environment remains stable throughout the global Hsp90β conformational cycle and highlighting a significant discrepancy with conformations observed in cryo-EM structures of the co-chaperone and client-bound complexes.