Molecular chaperones are a class of proteins that identify destabilized or misfolded proteins to prevent their aggregation as well as assist them in reaching their native conformations. Heat-shock protein 90 (Hsp90) proteins are a class of molecular chaperones of which Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog. While Grp94 is responsible for the folding and assembly of membrane and secretory proteins, it is researched for its role in the remodeling and activation of disease-causing proteins implicated in some cancers and neurodegenerative disorders. Grp94 is unique in that it contains a pre-N domain that is not conserved in other Hsp90 paralogs. In this study, we investigate Grp94’s pre-N domain by generating truncated pre-N domain constructs. Constructs were created through gene splicing and confirmed by sequencing. The proteins were expressed in BL21 cells and purified by chromatography. The proteins were confirmed to be >90% pure by SDS-PAGE. We conducted ATPase assays to determine the ATP hydrolysis rates of the full-length Grp94 and the truncation mutants. We used protein-protein interaction assays with biotinylated Grp94 to determine client binding abilities of the truncation mutants in comparison to full-length Grp94. The Grp94 pre-N truncation mutants have higher ATP hydrolysis rates than full-length Grp94. In contrast, the Grp94 truncation mutants exhibit similar binding with two client proteins (L2 and Δ131Δ). Therefore, we conclude that the pre-N domain of Grp94 has an inhibitory effect on ATP hydrolysis rates, but does not play a significant role in Grp94 client binding. This suggests that the pre-N domain may only play a structural role to enable Grp94 to assume an open conformation. Future work will further investigate Grp94’s pre-N domain by determining its ability to refold clients. Preston is hoping to one day research embryonic stem cell applications in healthcare and this work has provided him with immense experience around biological chemistry methods. Katherine will be pursuing a doctorate in Pharmacy and this work has provided her with hands-on experience and education in regards to performing biochemical methods and investigating chaperone proteins.
Authors: Preston Collins, Biochemistry Major and Katherine Vitou, Biochemistry and Premedical Studies Major
Advisor: Andrea Kravats, Department of Chemistry and Biochemistry
Graduate Advisor: Yaa Sarfowah Amankwah, Department of Chemistry and Biochemistry
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