Physical Chemistry of Cellular Liquid‐Phase Separation

Bentley, Emily P.; Frey, Benjamin B.; Deniz, Ashok A.

doi:10.1002/chem.201805093

Cited by 51 publications

(42 citation statements)

References 100 publications

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“…Phase separation of LAF-1 RGG is hypothesized to be driven by several different modes of interaction, including electrostatic, π-π, and cation-π interactions (27). In addition, hydrogen bonds and hydrophobic contacts may play a role in phase separation for sequences containing residues capable of such interactions (16,(33)(34)(35)(36)(37). However, it is difficult to characterize these interactions using experimental techniques due to the dynamic nature of the phase-separated proteins and the high spatiotemporal resolution needed to probe the interactions (35).…”

Section: Resultsmentioning

confidence: 99%

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

Schuster

Dignon

Tang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

235

302

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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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Section: Resultsmentioning

confidence: 99%

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

Schuster

Dignon

Tang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

235

302

View full text Add to dashboard Cite

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“…This protein has several interesting features that may be important in its function in SG formation. Intrinsically disordered regions (IDRs) of proteins are thought to play a major role in cellular LLPS by way of multivalent interactions (Banani et al, 2017;Bentley et al, 2019). In the case of G3BP, however, the story appears to be more nuanced.…”

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confidence: 99%

Networking and Dynamic Switches in Biological Condensates

Deniz

2020

Cell

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Cellular liquid-liquid phase separation (LLPS) plays a key role in the dynamics and function of RNA-protein condensates like stress granules. In this issue of Cell, Yang et al., Guillé n-Boixet et al., and Sanders et al. use a combination of experiment and modeling to provide an exciting mechanistic insight into the relationship between stress granules and LLPS, for example, in the context of protein disorder, switchable interactions, graph theory, and multiple interacting dense phases.

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“…Their functions include transcriptional regulation, cellular signaling, and dampening of the stress response. In addition, IDPs are key players in several neurodegenerative diseases (e.g., Parkinson's and Huntington's diseases), diabetes, and heart disease (Dunker, Bondos, Huang, & Oldfield, 2015; Wright & Dyson, 2015), as well as cellular compartmentalization by liquid‐liquid phase separation (Bentley, Frey, & Deniz, 2019).…”

Section: Commentarymentioning

confidence: 99%

Ratiometric Single‐Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins

Nasir

Bentley

Deniz

2020

CP Chemical Biology

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Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid‐liquid phase separation. The so‐called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein “structure‐function paradigm,” as their functions do not necessarily involve well‐defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single‐molecule fluorescence methods can overcome this ensemble‐average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single‐molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in‐depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel

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Physical Chemistry of Cellular Liquid‐Phase Separation

Cited by 51 publications

References 100 publications

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

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

Networking and Dynamic Switches in Biological Condensates

Ratiometric Single‐Molecule FRET Measurements to Probe Conformational Subpopulations of Intrinsically Disordered Proteins

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