Members of the 70-kD family of heat shock proteins (HSP70s) mediate protein-protein interactions, catalyzing protein folding, protein transport, the assembly of oligomeric proteins, and even the renaturation of heat-inactivated proteins-processes that are essential for life. Recent studies have revealed that HSP70s have a variety of important functions in the human immune system. Paradoxically, HSP70s are non- specific catalysts, that is, they appear to mediate protein-protein interactions by reacting in a relatively non-specific manner with unraveled segments of other proteins. The long-term objective of this research is to understand the molecular basis by which the heat shock-70 protein dnaK reacts non-specifically with other proteins, discriminating unfolded from folded proteins. Because the discrimination between folded and unfolded proteins is probably a kinetic effect, the specific aims to be pursued during this grant period involved elucidating the kinetics and mechanisms of three reactions-binding (P+dnaK-dnaK+P*), dissociation (dnaK-P*-dnaK+P) and ATP hydrolysis (nATP+dnaK+P*-nADP+dnaK+P), where P and P* represent non-fluorescent free labeled-peptide and fluorescent bound labeled-peptide (peptides are used to mimic the unraveled segments of other proteins).
The first aim i s to determine the rate-limiting step in the binding determine the mechanism of peptide dissociation from dnaK, to determine the effect of the peptide's structure on the rate of dissociation from dnaK, and to test the idea that there are multiple, overlapping interacting peptide binding site.
The third aim i s to understand how ATP-hydrolysis in the ATP-binding domain of dnaK is coupled to peptide dissociation from the peptide-binding domain.
The fourth aim i s to investigate the kinetics and mechanism of peptides binding to and dissociating from the peptide-binding domain fragment of dnaK, obtained by limited proteolysis of dnaK. The reactions between dnaK and fluorescently labeled-peptides will be followed using stopped- flow fluorescence spectrophotometry in conjunction with stopped-flow circular dichroism. Execution of these specific aims will provide quantitative information on the individual reaction steps involved in dnaK-catalyzed reactions.
