Sequence determinants of protein phase behavior from a coarse-grained model

Membraneless organelles important to intracellular compartmentalization have recently been shown to comprise assemblies of proteins which undergo liquid-liquid phase separation (LLPS). However, many proteins involved in this phase separation are at least partially disordered. The molecular mechanism and the sequence determinants of this process are challenging to determine experimentally owing to the disordered nature of the assemblies, motivating the use of theoretical and simulation methods. This work advances a computational framework for conducting simulations of LLPS with residue-level detail, and allows for the determination of phase diagrams and coexistence densities of proteins in the two phases. The model includes a short-range contact potential as well as a simplified treatment of electrostatic energy. Interaction parameters are optimized against experimentally determined radius of gyration data for multiple unfolded or intrinsically disordered proteins (IDPs). These models are applied to two systems which undergo LLPS: the low complexity domain of the RNA-binding protein FUS and the DEAD-box helicase protein LAF-1. We develop a novel simulation method to determine thermodynamic phase diagrams as a function of the total protein concentration and temperature. We show that the model is capable of capturing qualitative changes in the phase diagram due to phosphomimetic mutations of FUS and to the presence or absence of the large folded domain in LAF-1. We also explore the effects of chain-length, or multivalency, on the phase diagram, and obtain results consistent with Flory-Huggins theory for polymers. Most importantly, the methodology presented here is flexible so that it can be easily extended to other pair potentials, be used with other enhanced sampling methods, and may incorporate additional features for biological systems of interest.Author summary: Liquid liquid phase separation (LLPS) of low-complexity protein sequences has emerged as an important research topic due to its relevance to membraneless organelles and intracellular compartmentalization. However a molecular level understanding of LLPS cannot be easily obtained by experimental methods due to difficulty of determining structural properties of phase separated protein assemblies, and of choosing appropriate mutations. Here we advance a coarse-grained computational framework for accessing the long time scale phase separation process and for obtaining molecular details of LLPS, in conjunction with state of the art enhanced sampling methods. We are able to qualitatively capture the changes of the phase diagram due to specific mutations, inclusion of a folded domain, and variation of chain length. The model is flexible and can be used with different knowledge-based potential energy functions, as we demonstrate. We expect a wide application of the presented framework for advancing our understanding of the formation of liquid-like protein assemblies.