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

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

doi:10.1073/pnas.2000223117

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

Journal Article · Mon May 11 00:00:00 EDT 2020 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2000223117· OSTI ID:1618137

Schuster, Benjamin S.;

;

; Kelley, Fleurie M.;

; Jahnke, Craig N.; Simpkins, Alison G.; Regy, Roshan Mammen; Hammer, Daniel A.;

; Mittal, Jeetain

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.

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Research Organization:: Univ. of Pennsylvania, Philadelphia, PA (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES). Materials Sciences & Engineering Division; National Institutes of Health (NIH); National Science Foundation (NSF)

Contributing Organization:: University of Pennsylvania, Lehigh University, Rutgers University

Grant/Contract Number:: SC0013979; SC0007063; R01-NS116176; AC02-05CH11231

OSTI ID:: 1618137

Alternate ID(s):: OSTI ID: 1638215

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Vol. 117 Journal Issue: 21; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 119 works

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

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