Molecular Insights into Chaperone-Mediated Phase Separation and Amyloid Aggregation of Neuronal Intrinsically Disordered Proteins using Fluorescence Spectroscopy and Imaging

Abstract

Intrinsically disordered proteins (IDPs) are a special class of proteins that challenge the classical sequence-structure-function paradigm and exist as a dynamic, heterogeneous ensemble of rapidly interconverting conformations. While IDPs are involved in a myriad of critical physiological functions, their misfolding leads to the formation of amorphous aggregates or amyloids that are linked to various debilitating neurodegenerative disorders. The highly ordered amyloid assemblies share a common cross-β sheet conformation. Therefore, identifying the amyloid core residues or the specific regions of a protein that drive amyloid formation becomes crucial in developing targeted therapeutic strategies for amyloid related diseases. We developed an easy-to-use fluorescence-based methodology to map the amyloid core of a model IDP, alpha-synuclein, using tryptophan-induced quenching of thioflavin T (ThT). We believe that this distance-dependent Trp-induced quenching of ThT fluorescence via photoinduced electron transfer can be harnessed to obtain structural insights into disease-associated amyloid fibrils, surpassing the conventional application of ThT fluorescence that is limited to monitoring the in vitro aggregation kinetics. Increasing evidence suggests that in addition to the canonical membrane-enclosed organelles, cells contain a host of non-canonical membraneless organelles formed via intracellular phase separation of IDPs along with nucleic acids and other biomolecules. We demonstrate that a molecular chaperone tasked with cellular protein quality control can promote phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop), abolishing its abnormal conversion into solid-like pathological aggregates. Our study provides unique mechanistic insights into the phase separation-mediated chaperoning of proteins, preventing their transitions into pernicious, disease-causing, misfolded, and aggregated states of proteins. Such a chaperone-mediated mechanism can have a broader role in maintaining protein homeostasis and controlling the formation of toxic protein aggregates. We believe that the work described in this thesis can deepen our understanding of the role of protein quality control machinery in regulating the aberrant aggregation of proteins and the significance of tryptophan-induced quenching of ThT fluorescence to map the amyloid core architecture of the amyloidogenic IDPs.