Date of Award

Summer 2019

Project Type


Program or Major


Degree Name

Master of Science

First Advisor

Feixia Chu

Second Advisor

Xuanmao Chen

Third Advisor

Krisztina Varga


Heat shock protein 90 (Hsp90) is a highly conserved and dynamic molecular chaperone protein important for regulating protein activity and interactions in cellular signaling and adaptive stress responses. As a key regulatory protein, Hsp90 is often manipulated by cancer cells to enhance tumorigenic cell growth and even exists in some tumors as a multimeric complex called the “epichaperome.” With its linked role in cancer proliferation, Hsp90 serves as a promising cancer therapeutic target, with many Hsp90 inhibitors developed including geldanamycin and PU-H71. With limited information existing regarding epichaperome formation and structure, this study seeks to investigate potential regulatory mechanisms for epichaperome stabilization. In this study, we investigate the conformational states of Hsp90 targeted by geldanamycin and PU-H71 using chemical cross-linking and mass spectrometric analysis. Our data suggests that geldanamycin targets an open-like conformation of Hsp90, while PU-H71 targets a closed-like conformation of Hsp90 in a multicomplex epichaperome. Additionally, we detected enriched phosphorylation in the charged linker region of PU-H71-bound Hsp90 at residues S226 and S255. Mammalian expression vectors were developed containing mCherry linked to Hsp90. Within the Hsp90 sequence, S226 and S255 were mutated to phosphomimetic or phosphorylation-null amino acids. Confocal microscopy and immunoprecipitation analysis of mCherry linked to Hsp90 revealed that phosphorylation null mutants were associated much less with Hsp90 co-chaperones than unmutated Hsp90 and phosphomimetic mutated Hsp90. Overall, the results indicated that phosphorylation likely plays an important role in epichaperome formation and assembly.