The long term goals of the proposed research are to understand the functions and physiological roles of E. coli's hsp7O protein, DnaK, and its hsp6O protein, GroEL. These proteins are molecular chaperones and play roles in protein folding/unfolding and in the assembly/disassembly of protein complexes. Their expression is strongly induced by heat-shock and they play regulatory and functional roles in the heat-shock response as well as in metabolism at lower temperatures. A tremendous amount of information is being learned about hsp7O and hsp6O proteins from other organisms, particularly mammals and yeast and they are proving to be of medical interest both with respect to pathogenesis and autoimmune diseases. E. coli is a particularly attractive system for analyzing these highly evolutionarily-conserved proteins. We will carry out genetic analyses of DnaK function by analyzing mutations that permit the growth of dnaK mutants at elevated temperature, testing whether there is a cellular function that substitutes for DnaK at 30 degrees C, completing our analysis of the sidA1 mutation, and isolating mutations affecting the peptide-binding site of DnaK. We will characterize the in vivo and in vitro properties of mutant DnaK proteins altered at Thr199 by determining the phenotypes of dnak mutants with alterations of Thr199 and further characterizing the properties of DnaK proteins with alterations of Thr199. We will further characterize the temperature-dependence of the DnaK ATPase and autophosphorylation rates, explore the involvement of DnaK in the regulation of the heat shock response, and test the possibility that DnaK functions as a cellular thermometer that directly senses temperature. We will investigate the possible significance of phosphorylation in DnaK function by investigating whether other members of the hsp7O family undergo phosphorylation of the threonine corresponding to Thr199, examining the phosphorylation of DnaK in vivo, and testing whether phosphorylated DnaK preferentially binds to unfolded proteins. We will analyze the biochemical and physiological significance of the high molecular weight complexes containing DnaK that we have detected. We will analyze the physiological purpose and nature of the GroE-UmuC interaction and determine which element(s) of UmuC is responsible for its interactions with the GroE proteins.
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