Journal Article

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Kelley, Fleurie M., Ranganath, Aishwarya Kanchi, Jahnke, Craig N., Simpkins, Alison G., Regy, Roshan Mammen, Tang, Wai Shing, Dignon, Gregory L., Good, Matthew C., Mittal, Jeetain, Schuster, Benjamin S., Hammer, Daniel A.

2020

Overview

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

• Publisher: National Academy of Sciences
• Subject: Amino acid sequence / Amino Acid Substitution / Arginine / Arginine - chemistry / Biological Sciences / Biomimetics / Biophysics and Computational Biology / Caenorhabditis elegans Proteins - chemistry / Caenorhabditis elegans Proteins - genetics / Caenorhabditis elegans Proteins - metabolism / Chemistry / Computer simulation / Cytoplasm - metabolism / Domains / Hydrophobic and Hydrophilic Interactions / In vivo methods and tests / Interrogation / intrinsically disordered proteins / Intrinsically Disordered Proteins - chemistry / Intrinsically Disordered Proteins - genetics / Intrinsically Disordered Proteins - metabolism / Lipids / liquid-liquid phase separation / MATERIALS SCIENCE / Membrane proteins / membraneless / Microorganisms, Genetically-Modified / Molecular Dynamics Simulation / molecular simulations / Mutagenesis / Negative selection / Organelles / Phase separation / Phase Transition / Physical Sciences / Protein Domains / Proteins / recombinant proteins / Residues / RNA Helicases - chemistry / RNA Helicases - genetics / RNA Helicases - metabolism / Saccharomyces cerevisiae - genetics / Saccharomyces cerevisiae - metabolism / sequence determinants / simulation / Temperature / Tyrosine / Tyrosine - chemistry