MOLECULAR MECHANISM OF GROES/GROEL CHAPERONIN FUNCTION

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All organisms from bacteria to man respond to heat and other stresses that lead to an accumulation of unfolded polypeptides by rapidly increasing the synthesis of a small number of highly conserved, constitutively expressed gene products called heat-shock or stress- induced proteins. Evidence is rapidly accumulating that stress-induced proteins are normally involved in a diverse set of essential physiological processes, including efficient intracellular protein folding. The GroES and GroEL chaperonins are major heat-shock proteins from Escherichia coli that control protein folding in cells by regulating the release of unfolded polypeptide chains that are strongly bound to GroEL, in an ATP hydrolysis coupled reaction, to minimize their nonproductive aggregation. Despite the wealth of biochemical data that has led to descriptive models, little quantitative information is available to base a molecular mechanism for how these proteins facilitate refolding. The goal of this research is to gain insight into the molecular mechanism whereby GroES and GroEL assemble and catalyze efficient cellular protein folding by investigating the structural, kinetic and thermodynamic basis of their interaction with each other, and model peptides, and how these processes are coupled to ATP binding and hydrolysis. The interaction of GroES with GroEL will be characterized by sedimentation equilibrium using radiolabeled proteins to measure their strong association and to clarify the stoichiometry of their interaction in the presence of peptide and nucleotide effectors. The forces stabilizing polypeptide binding to GroEL will be described by titration calorimetry measurements of the association of ribonuclease S peptide to the chaperonin. A comparison of these energetic driving forces to the structure and interactions of the S peptide with the S protein will yield a thermodynamic description of the polypeptide binding site of GroEL, and may resolve apparent cooperative effects in polypeptide chain binding by GroEL. Rapid kinetic measurements of fluorescence changes upon peptide binding and dissociation, coupled with quench flow experiments to measure the rates of ATP binding, hydrolysis and product release, will elucidate how ATP binding is coupled to polypeptide chain release from GroEL. These experiments will provide the framework to test a simple working hypothesis for the regulation of the GroEL ATPase by GroES and peptide substrates. The potential to correlate structural changes measured by difference sedimentation velocity with functional perturbations will be exploited with engineered expression vectors to construct site-specific mutants in these essential gene products, in addition to enabling their purification from constitutive levels wild-type GroES and GroEL in a single step. Cysteine containing mutants will be constructed to prepare heavy atom derivatives to aid in solving the structures of the chaperonins from large, single crystals that diffract x rays to atomic resolution, enabling us to achieve our long range goal of correlating the molecular interactions of chaperone proteins with relevant structures along their reaction pathway.