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Phase separation processes facilitate the formation of membrane-less organelles and involve interactions within structured domains and intrinsically disordered regions (IDRs) in protein sequences. The literature suggests that the involvement of proteins in phase separation can be predicted from their sequences, leading to the development of over 30 computational predictors. We focused on intrinsic disorder due to its fundamental role in related diseases, and because recent analysis has shown that phase separation can be accurately predicted for structured proteins. We evaluated eight representative amino acid-level predictors of phase separation, capable of identifying phase-separating IDRs, using a well-annotated, low-similarity test dataset under two complementary evaluation scenarios. Several methods generate accurate predictions in the easier scenario that includes both structured and disordered sequences. However, we demonstrate that modern disorder predictors perform equally well in this scenario by effectively differentiating phase-separating IDRs from structured regions. In the second, more challenging scenario—considering only predictions in disordered regions—disorder predictors underperform, and most phase separation predictors produce only modestly accurate results. Moreover, some predictors are broadly biased to classify disordered residues as phase-separating, which results in low predictive performance in this scenario. Finally, we recommend PSPHunter as the most accurate tool for identifying phase-separating IDRs in both scenarios.
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Mechanosensitive ion channels MSL8, MSL9, and MSL10 have environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics in vitro
Abstract Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N‐terminal domains of MSL8, MSL9, and MSL10, three members of the MscS‐like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N‐termini of MSL channels are intrinsically disordered. We examined the N‐terminus of MSL10 (MSL10 N ) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10 N adopted a predominately helical structure when exposed to the helix‐inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10 N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8 N , MSL9 N , and MSL10 N have sharply differing propensities for self‐assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N‐termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment.
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- Award ID(s):
- 1929355
- PAR ID:
- 10465525
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 7
- Issue:
- 8
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/pld3.515
