Investigation of Dynamics along Divergent Tau Aggregation Pathways

Abstract

Intrinsically disordered proteins (IDPs) are ubiquitous and perform a range of functions vital to cell signaling, cytoskeletal architecture, and cellular physiology. IDP function depends on their ability to dynamically reconfigure themselves to interact with other proteins, lipids, and biopolymer binding partners. Microtubule-associated protein tau has been reported to form phase-separated droplets as well as a variety of amyloid fibril morphologies. This behavioral complexity makes tau an interesting model for studying the molecular recognition features, sequence-dependence, dynamic factors, and structural intricacies associated with IDP behaviors. Studies of tau in vitro rely on polyanions to induce aggregation, but recent evidence suggests that the morphology of fibrils formed in vitro is distinct from those formed in vivo and may be inducer dependent. While these observations indicate a tunable self-assembly landscape, we do not understand the structural or kinetic mechanisms by which a given inducer drives tau aggregation into one morphology or another. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments probe the accessibility of backbone amides throughout a protein and can track local secondary structure associated with tau self-assembly. However, the rapid fluctuations exhibited by tau and other IDPs can make them challenging to interrogate using conventional HDX-MS. We describe the application of a microfluidic mixing apparatus that extends the dynamic range of HDX-MS into the millisecond regime, revealing the conformational dynamics of monomeric tau. We also examine changes in HDX behavior observed over the course of tau aggregation reactions, complemented by fluorescence-based kinetic measurements. Taken together, these approaches could advance our understanding of the tau self-assembly landscape and demonstrate the value of HDX-MS in studying similarly challenging IDP systems.