Project Summary/Abstract Chaperone proteins are critical for cell survival and provide pathways for correct protein folding within the cell. Our research focuses on ATP-dependent chaperones in the endoplasmic reticulum, specifically Grp94. Grp94 belongs to the highly conserved Hsp90 superfamily, and like its cytosolic and mitochondrial paralogs, it folds and activates specific client proteins. Grp94 is of fundamental interest because it has limited mechanistic information compared to other paralogs. Grp94 is also unique because it has several observed structural and functional differences from paralogs that make it a promising drug target. Despite the importance of Grp94 chaperoning in protein homeostasis, the fundamental details of its chaperone cycle are not well characterized. Grp94 is of practical interest since aberrant protein folding in the endoplasmic reticulum results in medical issues such as type 2 diabetes, cancer, hepatitis B & C, and neurodegenerative and cardiovascular diseases. Understanding Grp94’s structure and function will provide a foundation for understanding how diseases caused by misfolded ER proteins can be treated and prevented. This fundamental knowledge of Grp94 mechanisms can aid in rational drug design. Current strategies involve ATP-competitive inhibitors that target all Hsp90 paralogs and inhibit both productive and unproductive chaperone activity, which results in toxicity. Novel therapeutic strategies include inhibiting chaperoning of toxic proteins while retaining chaperoning of non-toxic proteins and targeting specific Hsp90 paralogs; however, mechanistic information is required to move the field in this direction. Therefore, the studies of this chaperone mechanism will be of immense practical and economical value in the development of disease therapies. Understanding the conformational changes involved in the chaperone cycle is fundamentally important for identifying points of intervention. Understanding how client proteins and other chaperones structurally and functionally interact is fundamentally important for designing competitive modulators of Grp94. My lab will develop novel techniques for functional and structural studies of Grp94. We will tightly couple experimental and computational studies, which is a powerful combination of tools that will enable us to elucidate molecular details that wouldn’t be possible to obtain with either method individually. Using this approach, we will answer the following pertinent biological questions: (1) What are the preferred conformational states of Grp94 and which conformations co-exist in equilibrium? (2) How do cellular conditions and interactors influence Grp94’s conformational sampling? (3) What type of chaperone activity does Grp94 demonstrate and what are the requirements? (4) Where do client proteins interact on Grp94? (5) Are ATP hydrolysis events in the Grp94 dimer symmetric or asymmetric? The successful completion of these studies is expected to have an impor...