Molecular chaperoned comprises a diverse group of proteins which play important roles in assisting protein folding and assembly, in protein transport across membranes, and protein renaturation and/or prevention of aggregation as a result of various types of stress. A multi-faceted research program is proposed to investigate the function and structure of members of the approximately 70kDa (hsp79) class of chaperoned and their co-chaperones. The proposed studies focus on Hsc66 and Hsc20, novel chaperone and co-chaperone proteins encoded together with a (2Fe-2S) ferredoxin in a recently discovered operon in Escherichia coli. New findings suggest that Hsc66 and Hsc20 function in the folding and/or assembly of the ferredoxin and other iron-sulfur proteins in E. coli, and a combination of genetic and biochemical approaches are proposed to investigate the roles of Hsc66 and Hsc20 and the mechanism of chaperone-protein interactions. Systems for high level expression and for purification of Hsc66 and Hsc2 have been developed to allow characterization of the catalytic and peptide binding properties of the native proteins and of site-specific mutants. Structural studies on Hsc66 and Hsc20 are also proposed. Crystals of Hsc20 have been obtained, and x-ray diffraction data on the native protein (1.9A) and a heavy atom derivative (2.2A) have yielded a preliminary electron density map for modeling and structural refinement. Conditions for crystallization of Hsc66 are being developed. The results of these studies should lead to better general understanding of chaperone function and structure, and they should provide new insights into cellular mechanisms involved in the folding and assembly of iron-sulfur proteins and the regulation of these processes.
