Expanding the molecular language of protein liquid-liquid phase separation

Abstract

Understanding the relationship between an amino acid sequence and its phase separation has important implications for analyzing cellular function, treating disease, and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), leading to the development of a “molecular grammar” for LLPS. In this work, we further probed how sequence modulates phase separation and the material properties of the resulting condensates. Specifically, we used a model intrinsically disordered polypeptide composed of an 8-residue repeat unit and performed systematic sequence manipulations targeting sequence features previously overlooked in the literature. We generated sequences with no charged residues, high net charge, no glycine residues, or devoid of aromatic or arginine residues. We report that all but one of the twelve variants we designed undergo LLPS, albeit to different extents, despite significant differences in composition. These results support the hypothesis that multiple interactions between diverse residue pairs work in tandem to drive phase separation. Molecular simulations paint a picture of underlying molecular details involving various atomic interactions mediated by not just a handful of residue types, but by most residues. We characterized the changes to inter-residue contacts in all the sequence variants, thereby developing a more complete understanding of the contributions of sequence features such as net charge, hydrophobicity, and aromaticity to phase separation. Further, we find that all condensates formed behave like viscous fluids, despite large differences in their viscosities. The results presented in this study significantly advance the current sequence-phase behavior and sequence-material properties relationships to help interpret, model, and design protein assembly.

Sequence-Dependent Material Properties of Biomolecular Condensates and their Relation to Dilute Phase Conformations

“…We found near-constant η values at sufficiently low shear rates for all sequences, indicative of their Newtonian liquid-like behavior, similar to that seen for experimentally characterized disordered protein condensates. 18, 24, 32 Similar to the MSD, η depended on the degree of charge segregation within the sequence. To illustrate this dependence, we considered the zero-shear viscosity η 0 of all sequences, which is obtained as an average of the η values from the Newtonian plateau observed at low shear rates ( Fig.…”

Determinants of Disordered Protein Co-Assembly Into Discrete Condensed Phases

“…ask simply, to what extent is it possible for two IDRs to selectively assemble into distinct condensates with minimal co-mixing in the absence of shared ligands or macromolecules? Recently, our lab and others have revealed an amino acid code governing homotypic interactions that underlie liquid-liquid phase separation (LLPS) of various IDRs [18][19][20][21][22][23] . Coarse-grained models have provided insights on key residues or motifs that influence IDR saturation concentrations (C sat ) and client partitioning to condensed phases.…”

“…Recently, our lab and others have revealed an amino acid code governing homotypic interactions that underlie liquid-liquid phase separation (LLPS) of various IDRs [18][19][20][21][22][23] . Coarse-grained models have provided insights on key residues or motifs that influence IDR saturation concentrations (C sat ) and client partitioning to condensed phases.…”

Sequence-Encoded Differences in Phase Separation Enable Formation of Resilin-like Polypeptide-Based Microstructured Hydrogels

“…In recent reports from our laboratories, Rekhi et al showed that decreasing the aromatic character of similar resilin-derived model artificial intrinsically disordered proteins (A-IDPs) capable of undergoing UCST behavior led to fewer interchain contacts in the protein condensates. 59 In the current studies, as indicated by their opacity, it is possible that a higher extent of interchain contacts and polymerization-induced phase separation (PIPS) 60,61 in bulk Y-4Ac hydrogels afforded them higher shear storage moduli compared to L-4Ac bulk hydrogels of the same composition (Figures S14−S16). Compared to a nonphase-separated hydrogel, local enrichment of the polypeptide likely results in formation of bicontinuous macronetworks comprising cross-linked bundles of polypeptide chains interconnected within a solvent-rich phase (Figure S17).…”