Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration-and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.Oxidative stress generated by the production of reactive oxygen species is an inevitable consequence of aerobic life. In addition, the extracellular environment can also cause oxidative stress to the cells (1, 2). Heat shock is shown to increase oxidative stress (3). Oxidative stress, inter alia, results in misfolding and aggregation of several cellular proteins. In order to combat oxidative stress, cells produce proteins that detoxify reactive oxygen species and repair harmful changes. The Escherichia coli cytoplasm, like that of most other organisms, is largely reducing in nature although under oxidative stress, the redox status of the cell changes significantly. Several cellular proteins are regulated by redox-dependent post-translational modifications (4).Until recently, no molecular chaperone was known that could be turned on or off by a post-translational event acting as a switch. Jakob et al. (5) showed that a member of the highly conserved cytoplasmic prokaryotic molecular chaperones, Hsp33, is regulated by the redox state of the medium. Under reducing conditions, Hsp33 is inactive. Hsp33 has four absolutely conserved cysteine residues: Cys-232, -234, -265, and -268. In the reduced state, they are coordinated to a zinc atom (6). Oxidizing conditions lead to the release of zinc and formation of two intramolecular disulfide bonds, Cys-232 with Cys-234 and Cys-265 with Cys-268 (7). Upon such oxidative activation, Hsp33 binds to aggregation-prone states of proteins, preventing their aggregation and acting as...