A generic approach to study the kinetics of liquid–liquid phase separation under near-native conditions

Lindt, Joris Van; Bratek‐Skicki, Anna; Nguyen, Phuong Nga; Pakravan, Donya; Durán-Armenta, Luis F.; Tantos, Ágnes; Pancsa, Rita; Bosch, Ludo Van Den; Maes, Dominique; Tompa, Péter

doi:10.1038/s42003-020-01596-8

Cited by 48 publications

(77 citation statements)

References 28 publications

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“…Assessing pH-dependent disorder is also valuable for optimizing solvent conditions and to speed up buffer screenings. In addition, this tool can be of help in the study of pH-modulated liquid–liquid phase separation (LLPS), where pH-jumps are used to prevent or trigger LLPS, allowing kinetic analysis in near-native conditions [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

DispHScan: A Multi-Sequence Web Tool for Predicting Protein Disorder as a Function of pH

et al. 2021

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Proteins are exposed to fluctuating environmental conditions in their cellular context and during their biotechnological production. Disordered regions are susceptible to these fluctuations and may experience solvent-dependent conformational switches that affect their local dynamism and activity. In a recent study, we modeled the influence of pH in the conformational state of IDPs by exploiting a charge–hydrophobicity diagram that considered the effect of solution pH on both variables. However, it was not possible to predict context-dependent transitions for multiple sequences, precluding proteome-wide analysis or the screening of collections of mutants. In this article, we present DispHScan, the first computational tool dedicated to predicting pH-induced disorder–order transitions in large protein datasets. The DispHScan web server allows the users to run pH-dependent disorder predictions of multiple sequences and identify context-dependent conformational transitions. It might provide new insights on the role of pH-modulated conditional disorder in the physiology and pathology of different organisms. The DispHScan web server is freely available for academic users, it is platform-independent and does not require previous registration.

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Section: Discussionmentioning

confidence: 99%

DispHScan: A Multi-Sequence Web Tool for Predicting Protein Disorder as a Function of pH

et al. 2021

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“…The kinetics of these processes, and the effect of different conditions or additives on these kinetics, is of particular interest. 14,15 However, these processes are di cult to study by existing techniques. Light scattering due to the presence of liquid droplets, or uctuations in density or refractive index often results in opalescent or turbid solutions, rendering quantitative optical approaches challenging.…”

Section: Introductionmentioning

confidence: 99%

“…16,17 Fluorescence microscopy using labelled LLPS components or dyes may report on the radius of droplets, but not the concentration of the two phases. 15,18 Additionally, layer separation adds a complicating spatial component, due to non-uniform distribution of the phases throughout the sample. 19 Therefore, the physical and geometrical constraints of biophysical techniques mean each may be limited to studying one aspect of LLPS, and further characterisation techniques are needed to reach a more holistic assessment, particularly regarding the evolution of the concentration and volumes of the two phases.…”

Section: Introductionmentioning

confidence: 99%

Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy

Bramham

Golovanov

2022

Preprint

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Liquid-liquid phase separation (LLPS) of protein solutions is increasingly recognised as an important phenomenon in cell biology and biotechnology. However, opalescence and concentration fluctuations render LLPS difficult to study, particularly when characterising the kinetics of the phase transition and layer separation. Here, we demonstrate the use of a probe molecule trifluoroethanol (TFE) to characterise the kinetics of protein LLPS by NMR spectroscopy. The chemical shift and linewidth of the probe molecule are sensitive to local protein concentration, with this sensitivity resulting in different characteristic signals arising from the dense and lean phases. Monitoring of these probe signals by conventional bulk-detection 19F NMR reports on the formation and evolution of both phases throughout the sample, including their concentrations and volumes. Meanwhile, spatially-selective 19F NMR, in which spectra are recorded from smaller slices of the sample, was used to track the distribution of the different phases during layer separation. This experimental strategy enables comprehensive characterisation of the process and kinetics of LLPS, and may be useful to study phase separation in protein systems as a function of their environment.

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“…In particular, the cooperation between long polymers, such as RNAs, together with folded proteins and intrinsically disordered proteins (IDPs) may be an essential feature of many condensates [3,39,40]. Furthermore, constituent binding affinity, valency, liquid network connectivity, and critical post-translational modifications all play a role in regulating BCs [41][42][43][44][45][46][47][48].…”

Section: Introductionmentioning

confidence: 99%

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation

Lyonnais

Sadiq

Lorca-Oró

et al. 2021

Viruses

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A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

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A generic approach to study the kinetics of liquid–liquid phase separation under near-native conditions

Cited by 48 publications

References 28 publications

DispHScan: A Multi-Sequence Web Tool for Predicting Protein Disorder as a Function of pH

DispHScan: A Multi-Sequence Web Tool for Predicting Protein Disorder as a Function of pH

Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy

The HIV-1 Nucleocapsid Regulates Its Own Condensation by Phase-Separated Activity-Enhancing Sequestration of the Viral Protease during Maturation

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