DESCRIPTION: Molecular chaperones are proteins that interact with nascent proteins, denatured proteins or other macromolecular assemblies and help them achieve native tertiary or quaternary structure. The prokaryotic molecular chaperones DnaK, DnaJ and GrpE cooperate to sequester aggregation-sensitive nascent polypeptides and unfolded proteins in an ATP-dependent manner, effectively removing them from the crowded cellular environment. The recent X-ray crystal structure of the DnaK ATP-hydrolyzing (ATPase) domain complexed with the nucleotide exchange factor GrpE revealed that GrpE induces a nucleotide-complex destabilizing conformational change in the ADP-bound form o DnaK. GrpE is a very elongated, cruciform-shaped molecule that causes the deep nucleotide binding cleft of the DnaK ATPase domain (which resembles actin) to be opened up. In its role as a nucleotide exchange factor, the action of GrpE is related conceptually to that of other exchange factors that operate in a variety of cellular timing mechanisms. Some issues highlighted by the GrpE-DnaK-ATPase complex are fundamental questions that can be asked of all nucleotide exchange factors, such as how GrpE discriminates between the ADP-and the ATP-bound forms of DnaK, and how GrpE is displaced from DnaK by the binding of ATP. This proposal is concerned with structural and functional aspects of nucleotide exchange that govern the DnaK chaperone cycle. The specific aims of this proposal are to: 1) Determine how GrpE recognizes the ADP-state of DnaK, but binds and stabilizes the stereochemically non-equivalen nucleotide-free DnaK by alanine-scanning mutagenesis of the GrpE-DnaK interfac and X-ray crystallographic studies of nucleotide-bound DnaK. 2) Study the mechanism of displacement of GrpE from DnaK by exploiting the asymmetric natur of GrpE when bound to DnaK, and testing with an in vitro protein folding assay 3) Investigate the allosteric communication between DnaK ATPase domain and peptide binding domain via GrpE by X-ray crystallography and mutagenesis of th substrate-dissociating region of GrpE. The long-term goals of this proposal ar to understand the function of nucleotide exchange factors in a structural context, and to further structural knowledge of the molecular chaperones.