This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hsp70 proteins mediate trafficking, folding and refolding of proteins in all known cellular structures. The protein is 70 kDa, but has a three-domain structure: ATPase domain (44 kDa) substrate-binding domain (15-20 kDa) and C-terminal domain (15-20 kDa). The chaperone binds to unfolded proteins and switches its affinity for these substrates by binding to ATP through an allosteric mechanism. The domains as well as constructs containg both domains can be individually expressed and folded. We have published structures on the substrate binding domain, the nucleotide binding domain, and have recently defined a global structure for a 54 kDa construct containing both domains. The current experiments serve to define the interface section between the domains, with NOE's. The protein is 13C, 15N, 2H and 13C1H3 labeled.