Single-Chain Dimensions of an Archetypal Phase-Separating Intrinsically Disordered Protein Region

Abstract

Membrane-less organelles (MLOs) are non-stoichiometric assemblies of concentrated biomolecules lacking a surrounding lipid membrane and form via liquid-liquid phase separation (LLPS). They facilitate a variety of cellular processes, and the dysregulation of phase separation can lead to debilitating diseases. Intrinsically disordered regions (IDRs) in proteins have often been found to drive phase separation. Due to lack of structure, disordered proteins are challenging to study with traditional structural biology methods, and this combined with crowded dense phase environments requires careful experimental design in order to extract quantitative information. We are directing our studies at an archetypal IDR that undergoes homotypic phase separation, i.e. the prion-like domain (PLD) of hnRNPA1. Here we use single-molecule fluorescence techniques to explore the dimensions and dynamics of hnRNPA1 in light and dense phases. We characterize single-chain behavior in response to salt concentration, which is used to induce phase separation, and elucidate how this is modulated by sequence variation. Initial measurements show that the PLD is a compact disordered protein in the dilute regime and that modulation of the driving force for phase separation also modulates the single-chain dimensions. This work will provide insights into structural and dynamic features of phase-separating biomolecules and will enable the investigation of maturation effects responsible for disease states.