Conformational Fingerprinting of Phase Separation and Amyloid Formation Through the Lens of Vibrational Raman Spectroscopy

Abstract

Intrinsically disordered proteins (IDPs) are a special class of proteins that confronts the newlineclassical sequence-structure-function paradigm and exists as a dynamic, heterogeneous newlineensemble of rapidly interconverting conformations. While IDPs are involved in a myriad of newlinecritical physiological functions, their misfolding leads to the formation of amorphous newlineaggregates or amyloids that are linked to various debilitating neurodegenerative disorders. The newlinehighly ordered amyloid assemblies share a common core architecture exhibiting a structural newlinediversity in their supramolecular packing arrangement within the backbone. Such an altered newlinepacking results in amyloid polymorphism that is often responsible for distinct amyloid strains. newlineWe utilized vibrational Raman spectroscopy coupled with hydrogen/deuterium exchange to newlinestructurally distinguish distinct amyloid polymorphs displaying altered hydrogen bonding newlineability and supramolecular packing within the cross-and#946; structural motif. Such structural newlineinvestigations are crucial to discern the mechanism of amyloid polymorphism and the newlinestructure-pathology relationship. Increasing evidence has suggested that in addition to the newlinecanonical membrane-bound organelles, cells contain a host of non-canonical membrane-less newlineorganelles formed via intracellular phase separation of IDPs along with nucleic acids and other newlinebiomolecules. These biomolecular condensates are involved in various cellular functions and newlinehuman pathologies. We developed and adapted a highly sensitive, single-droplet structural tool newlineinvolving dispersive laser Raman spectroscopy in a microscopy format that offers a wealth of newlinefundamental molecular information within the condensed phase. Our novel single-droplet newlinesurface-enhanced Raman scattering (SERS) technique using plasmonic nanostructures newlineilluminate the inner workings of the protein droplets and captures the crucial interactions, newlineconformational heterogeneity, and structural distribution in a single droplet fashion. Taken newlinetogether, this thesis elucidates the utility of vibrational