The Hsp90 family of chaperones functions to target misfolded proteins, prevent aggregate formation and bind to specific substrates for proper folding. Paralogs of the Hsp90 family exist in the cytoplasm (Hsp90), in the mitochondria (TRAP1) and in the endoplasmic reticulum (Grp94). The ER paralog Grp94 is of particular interest to this investigation since it is understudied and is implicated in a variety of diseases, including cardiovascular disease, neurodegenerative diseases, and cancer. Grp94 is a homodimer composed of an N-terminal domain (NTD), a charged linker region, the middle domain (MD), and the C-terminal domain (CTD). Depending on cellular conditions, Grp94 can act as a holdase to prevent protein aggregation or as a foldase to directly remodel clients. Previous research in our lab investigated the prevention of aggregation activity of Grp94. Studies with individual Grp94 domains indicated that model misfolded clients bind at the NTD of Grp94. Previous literature identified the site His146 on the NTD that significantly reduced peptide binding in comparison to wild-type (WT) Grp94. Interestingly, the peptide binding mutant located in the NTD, H146D, resulted in decreased aggregation of both substrates, possibly due to increased electrostatic interactions between the charged residue and substrate. This was an interesting finding, because client remodeling and peptide binding were thought to be distinct and occur in different regions of Grp94. In this work we investigate how point mutations in the NTD of Grp94 affect substrate binding using fluorescently labeled peptides. We have created various mutants within the general peptide binding region of Grp94. We are assessing peptide binding affinity with WT Grp94 and mutants. Together, this information will provide insight into charged interactions between client and chaperone and enable us to understand how Grp94’s NTD functions in binding potentially harmful protein aggregates.
Author(s): Kylie Paul, Erin Unruh, Nancy Rotich, Audrey McCamish, and Andrea Kravats, Ph.D
Advisor(s): Andrea Kravats, Department of Chemistry and Biochemistry
Erin Unruh, Department of Chemistry and Biochemistry
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