Mammalian (and other) cells respond to stress by increasing expression of a class of proteins known as heat shock proteins. Most of these proteins are produced constitutively under normal conditions and play essential roles in the life of the cell. Recent studies have shown that the mammalian hsp 70 family of heatshock proteins, and related polypeptide chain binding proteins and molecular chaperones (e.g. GroEL) from other sources, can bind to short peptides, and to nascent polypeptides in cells, and are involved in various aspects of protein assembly, translocation and folding. The relatively large fraction of cell protein, especially under conditions of stress, present as this type of protein, and the potential lethality of mutant forms, indicates their critical importance to cell function. Even though it is clear that proteins fold spontaneously in vitro, it is not clear that this is the case in vivo. In fact, a good case can be made for the need for some form of assistance in folding and assembly in the cell in order to minimize competing side reactions. The goal of the proposed research is to determine the molecular details of how hsp 70 and related polypeptide chain binding proteins facilitate the folding and assembly of substrate proteins. Biophysical characterization of the hsp 70 proteins will provide information on their structure and stability. A major part of the proposal concerns elucidation of the molecular details of the interaction of the hsp 70 with its substrate protein, and the role of ATP in this process. These studies will be carried out under in vitro conditions. The stoichiometry and binding constants for the interactions will be determined. A range of proteins from small monomeric to large oligomeric will be used as substrate proteins. The effect of hsp 70 on the kinetics of protein folding, and the affinity of hsp 70 for intermediates in folding will be determined. The structural and sequence specificity requirements for hsp 70 binding to proteins will be ascertained. From these investigations we expect to be able to provide a detailed molecular description of the function of this important class of cellular agent.
| Palleros, D R; Reid, K L; Shi, L et al. (1993) DnaK ATPase activity revisited. FEBS Lett 336:124-8 |
| Palleros, D R; Shi, L; Reid, K L et al. (1993) Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate. Biochemistry 32:4314-21 |
