Molecular Horizons PhD Exit Seminar - Nicholas Marzano

Protein misfolding and aggregation are associated with the pathogenesis of a wide range of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. The regulation of protein homeostasis is reliant on the Hsp40/Hsp70 system of molecular chaperones, which play critical roles in de novo protein folding and the refolding of misfolded proteins. However, questions remain regarding the precise molecular mechanisms by which Hsp70 remodel the client and how this contributes towards efficient folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes of individual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system.

For the first time, we observed multiple cycles of chaperone binding-and-release to an individual client during refolding. Furthermore, we demonstrate that Hsp40 remodels misfolded proteins via a conformational selection mechanism whereas Hsp70 resolves misfolded states via mechanical unfolding. Additionally, this work characterized a novel dye, called ASCP, that can be used as an alternative to thioflavin T to monitor the fibrillar aggregation of disease-associated proteins. ASCP also possesses an enhanced Stokes shift that could be utilized in single-laser two-color TIRF microscopy experiments to visualize the binding of molecular chaperones to amyloid fibril aggregates.