PROJECT SUMMARY/ABSTRACT The mitochondrial heat shock protein (Hsp) 60 molecular chaperone is critical in proteostasis and stress response, catalyzing ATP hydrolysis-dependent folding of mitochondrial proteins. While the precise folding mechanism is unknown, Hsp60 is proposed to function like the bacterial ortholog GroEL in which unfolded `client' proteins are enclosed in its central chamber, formed in part by its co-chaperone Hsp10, allowing them to fold without interference from other cellular components. Mutations in Hsp60 cause severe neurodegenerative diseases termed hereditary spastic paraplegias, and Hsp60 expression is upregulated in a subset of cancers, making this chaperone a significant therapeutic target. However, high-resolution structural information on Hsp60 is limited, leaving unresolved critical questions about oligomer organization, allosteric regulation, and interactions with client proteins throughout its reaction cycle. The objective of this proposal is to determine the mechanism of Hsp60 function using an integrated structural and chemical biology approach. Herein, I propose to solve high- resolution cryo-electron microscopy (cryo-EM) structures of Hsp60-client complexes at different nucleotide- bound and oligomeric states, in order to identify client interaction surfaces important for Hsp60-assisted protein folding (SA1). In exciting preliminary data, I have generated high-resolution (2.5 Å) cryo-EM structures of the ATP-bound Hsp60-Hsp10 double ring complex. This structure reveals novel inter-ring arrangements, indicating a distinct chaperonin mechanism of action and providing an excellent basis for the proposed studies. To complement these studies, I will also investigate the effects of disease-causing mutations and small molecule inhibitors on Hsp60 structure and function, in order to identify elements critical for Hsp60 function, and determine how this complex macromolecular machine can be perturbed (SA2). This will be accomplished by using biochemical and biophysical assays, as well as by solving cryo-EM structures of inhibitor-bound and mutant Hsp60 complexes. These studies will dramatically increase understanding of Hsp60 mechanism, as well as contribute to the development of the first well-validated Hsp60 chemical probes. This work, and my concomitant development as a structural and chemical biologist, will be enabled by a unique training environment formed by the labs of Dan Southworth and Jason Gestwicki, experts in studying molecular chaperones using cryo-EM and chemical biology, respectively.