Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 21, Pages 11421-11431Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2000223117
Keywords
liquid-liquid phase separation; membraneless organelles; molecular simulations
Categories
Funding
- US Department of Energy, Office of Science, Basic Energy Sciences awards [DE-SC0007063, DE-SC0013979]
- NIH [R01-NS116176, F32-GM119430, R01-EB028320]
- NSF [TG-MCB-120014, 1845734]
- National Energy Research Scientific Computing Center - Office of Science of the US Department of Energy [DE-AC02-05CH11231]
- Burroughs Wellcome Fund
- U.S. Department of Energy (DOE) [DE-SC0013979] Funding Source: U.S. Department of Energy (DOE)
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1845734] Funding Source: National Science Foundation
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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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