Studying the Functional Mechanism of the Heat Shock Protein 90

Hsp90 is an essential molecular chaperone in eukaryotic cells that supports the stability and function of numerous proteins critical for cellular health. This thesis focuses on enhancing the mechanistic understanding of Hsp90, particularly its interactions with co-chaperones and the regulation of client proteins. Using advanced single-molecule techniques such as fluorescence microscopy and optical tweezers, we discovered that the binding stoichiometry of the co-chaperone Aha1 to Hsp90 functions as an additional regulatory mechanism, which has likely been underestimated in the past and may also play a role in interactions with other co-chaperones. This work also presents a novel method for generating protein constructs that integrate fluorescence microscopy with optical tweezers in protein studies, addressing the limitations of traditional force spectroscopy, which typically probes in only one direction and may miss critical conformational changes. Furthermore, the defining characteristics of Hsp90 client proteins are investigated, focusing on leucine-rich repeat kinase 2 (LRRK2) and Hsp90's role in regulating its function. Compared to wild-type LRRK2, a Parkinson's disease-associated mutant LRRK2 exhibits increased binding affinity to a Hsp90-co-chaperone linker protein, potentially due to a shift in the monomer-dimer equilibrium towards the monomeric state of LRRK2. This finding may also apply to other Hsp90 client kinases whose activation is regulated by dimerisation. Disruption of the Hsp90-co-chaperone complex in a cellular environment impairs LRRK2 localisation and kinase activity. In summary, this research enhances our understanding of Hsp90’s functional mechanisms, paving the way for targeted drug development to treat conditions such as cancer and neurodegenerative disorders.