This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Molecular chaperones play essential roles in cellular maintenance and response to stress by protecting unfolded and misfolded proteins until they can be folded or assembled into complexes and then helping promote successful folding and assembly. They also partner with degradation machinery to manage cellular 'quality control'and prevent accumulation of potentially deleterious misfolded products. Chaperones work together to form a complex proteostasis network that is essential for cell survival. The ubiquitous Hsp70 protein family of chaperones prevents incorrect interactions in unfolded proteins that can lead to misfolding or aggregation by cycles of binding and release of client proteins regulated by ATP hydrolysis and further modulated by co-chaperones such as nucleotide exchange factors (NEFs) and Hsp40s. Understanding cellular roles of Hsp70s requires in-depth elucidation of the Hsp70 allosteric mechanism and interactions with co-chaperones;this is essential in order to develop therapeutic strategies based on modulation of Hsp70 chaperones. The goal of our work at the ACERT facility is to obtain crucial information for a full description of the structural ensembles of the E. coli Hsp70, DnaK, throughout its functional allosteric cycle and upon interaction with co-chaperones. We will determine distance profiles between various spin pairs strategically located throughout the protein. These distance profiles will provide information about the structures populated by DnaK in various nucleotide- or substrate- or co-chaperone-bound states. In addition, we will use double electron-electron resonance (DEER) experiments to identify sparsely populated conformers within these ensembles. These profiles, in conjunction with paramagnetic relaxation enhancement NMR data being collected at U. Mass will enable us to map out the allosteric landscape of an Hsp70.
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