Balancing stability, dynamics and kinetics in phase separation of intrinsically disordered proteins

Abstract

Liquid-liquid phase separation is a ubiquitous molecular phe-nomenon that plays crucial roles in a multitude of essential cellular activities. Intrinsically disordered proteins (IDPs), which lack well-defined three-dimensional structures, are prevalent participants in phase separation due to their inherent potential for promoting multivalent binding-the major driving force for this process. Understanding the underlying mechanisms of phase separation is challenging, as phase separation is a complex process, involving numerous molecules and various types of interactions. Here, we used a simplified coarse-grained model of IDPs to investigate the thermodynamic stability of the dense phase, conformational properties of IDPs, chain dynamics and kinetic rates of forming condensates. We focused on the IDP system, in which the oppositely charged IDPs are maximally segregated, inherently possessing a high propensity for phase separation. By varying interaction strengths, salt concentrations and temperatures, we observed that IDPs in the dense phase exhibited highly conserved conformational characteristics, which are more extended than those in the dilute phase. This implies that condensate formation acts as a protective shield, enabling IDPs to maintain conformational ensemble with high resistance to the changes in interactions and environmental conditions. Although the chain motions and global conformational dynamics of IDPs in the condensates are slow due to the high viscosity, local chain flexibility at the short timescales is largely preserved with respect to that at the free state. Strikingly, we observed a non-monotonic relationship between interaction strengths and kinetic rates for forming condensates. As strong interactions of IDPs result in high stable condensates, our results suggest that the thermodynamics and kinetics of phase separation are decoupled and optimized by the speed-stability balance through underlying molecular interactions. Our findings contribute to the molecular-level understanding of phase separation and offer valuable insights into the developments of engineering strategies for precise regulation of biomolecular condensates.