We propose to continue our studies aimed at elucidating the mechanisms by which the E. coli DnaK (Hsp70), DnaJ (Hsp40), and GrpE heat shock proteins cooperatively function as molecular chaperones. Homologues of these highly conserved Hsp70 and Hsp40 chaperones play vital roles in cells of all living organisms through their action in many aspects of protein metabolism, including protein folding, cellular growth control, neurotransmission, steroid receptor maturation, tumor suppression, and oncogenesis. We will continue our ongoing kinetic analysis of the intrinsic ATPase of DnaK with the goal of exploring how the coupling of the ATPase cycle to the binding and release of polypeptide substrates is affected by (i) wild type and mutant DnaJ cochaperones, (ii) the affinity of nucleotides for DnaK, or (iii) small molecule effectors such as inorganic phosphate and monovalent cations. In a parallel approach, we will construct a series of site-specific DnaK and DnaJ substitution mutants that will have single tryptophan residues inserted at various strategic locations. These locations will be chosen with the anticipation that the fluorescence properties of the substituted tryptophan will be sensitive to structural or conformational changes that occur during interaction of DnaK with DnaJ, GrpE, or nucleotides or during interaction of either DnaJ or DnaK with polypeptide substrates. Fluorescence changes occurring in these substituted proteins will be monitored by stopped-flow kinetics, which will facilitate detailed analysis of individual steps in the chaperone reaction cycle. We will also use surface plasmon resonance technologies to characterize further the interaction of DnaK with wild type and mutant forms of DnaJ and to probe the mechanisms involved in the selection of polypeptide substrates for DnaK. Finally, we will investigate the molecular mechanisms involved in a prototypical chaperone-mediated protein remodeling reaction. Specifically, we shall study the DnaJ- and DnaK-mediated disassembly of a highly stable nucleoprotein complex, formed on short single-stranded oligonucleotides, that contains the bacteriophage lambda O and P replication proteins and the E. coli DnaB helicase. Radiolabeled proteins and transient kinetic assays will be used to more precisely describe the nature of reactants, intermediates, and disassembly products.