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

The biological functions of the molecular chaperone Hsp90 are driven by pronounced conformational dynamics, yet the local structural features of these dynamics under phyisological conditions remain incompletely understood. Here, we systematically assessed the solvent accessibility of native cysteine residues in wild-type human Hsp90β in the apo state using a panel of thiol-reactive chemical probes with distinct physicochemical properties. Mass spectrometry identifies Cys366 as the most preferentially modified native cysteine in solution. Notably, the chemical accessibility of Cys366 is not measurably affected by nucleotide binding or hydrolysis, indicating that nucleotide-induced shifts in the Hsp90β conformational ensemble do not significantly impact the local environment close to this site. These results highlight the value of a practical framework for selecting and interpreting cysteine labeling sites in Hsp90 and related chaperone systems and also indicate a strategy for probing local accessibility of structural segments in conformationally dynamic protein complexes.