Liquid-liquid phase separation and aggregation of a globular folded protein SUMO1

Abstract

Many studies in recent years have investigated the phenomenon of liquid-liquid phase separation (LLPS) in proteins. LLPS is reported in intrinsically disordered proteins (IDPs) or in proteins with intrinsically disordered regions (IDRs) lacking a well-defined three-dimensional structure. However, the occurrence of LLPS in folded proteins, that lacks IDRs is not widely known. It is generally assumed that the compact structure and limited flexibility of folded proteins hinder their ability to establish weak and dynamic interactions crucial for LLPS. Contrary to the prevailing understanding, we present direct evidence of rapid phase separation of a globular protein, SUMO1, occurring under crowded conditions at physiological pH and room temperature. The protein molecules in the liquid droplets undergo conformational changes with time, monitored by intrinsic tryptophan fluorescence. We also demonstrate the phase transition of SUMO1 droplets from liquid to solid state with maturation, ultimately leading to aggregation. The SUMO1 aggregates contain a significant amount of beta-sheet structure but have amorphous morphology, probed by several spectroscopic techniques (Thioflavin T fluorescence, Raman Spectroscopy, and TEM). Our findings provide insights into the behaviour of SUMO1 protein in crowded environment, albeit, the underlying mechanism is not well understood and may differ from those of IDPs. Furthermore, SUMO1 protein is known to colocalize in inclusion bodies of IDPs, and therefore, LLPS of SUMO1 may have important biological implications in neurodegenerative diseases.