Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone

The heat shock protein Hsp33 is a very potent molecular chaperone with a di stinctive mode of functional regulation; its activity is redox-regulated. I n its reduced form all six cysteinyl residues of Hsp33 are present as thiol s, and Hsp33 displays no folding helper activity. Exposure of Hsp33 to oxid izing conditions like H2O2, however, rapidly converts Hsp33 into an efficie nt molecular chaperone. Activated Hsp33 binds tightly to refolding intermed iates of chemically denatured luciferase and suppresses efficiently their a ggregation in vitro, Matrix-assisted laser desorption/ionization-mass spect rometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accom panied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(252)-S-S-Cys(234) and Cys(265)-S-S-Cys(268). Cys(141), although not in volved in disulfide bond formation, was found highly reactive toward chemic al modifications. In contrast, Cys(239) is readily accessible under reducin g conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond struc ture, as a result of changes in the cellular redox potential, results in th e activation of a molecular chaperone.