A Novel Coarse-Grained Model to Study Liquid-Liquid Phase Separation of Disordered Proteins

Phase separation of intrinsically disordered proteins is important for the formation of membraneless organelles, or biomolecular condensates, which play key roles in the regulation of biochemical processes within cells. In this work, we investigated the phase separation of different sequences of a coarse-grained model for intrinsically disordered proteins and discovered a surprisingly rich phase behavior. We studied both the fraction of total hydrophobic parts and the distribution of hydrophobic parts. Not surprisingly, sequences with larger hydrophobic fractions showed conventional liquid-liquid phase separation. The location of the critical point was systematically influenced by the terminal beads of the sequence, due to changes in interfacial composition and tension. For sequences with lower hydrophobicity, we observed not only conventional liquid-liquid phase separation, but also reentrant phase behavior, in which the liquid phase density decreases at lower temperatures. For some sequences, we observed formation of open phases consisting of aggregates, rather than a normal liquid. These aggregates had overall lower densities than the conventional liquid phases, and exhibited complex geometries with large interconnected string-like or membrane-like clusters. Our findings suggest that minor alterations in the ordering of residues may lead to large changes in the phase behavior of the protein, a fact of significant potential relevance for biology.

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Structure of biomolecular condensates from dissipative particle dynamics simulations

10.1101/2019.12.11.873133 ◽

2019 ◽

Author(s):

Julian C. Shillcock ◽

Maelick Brochut ◽

Etienne Chénais ◽

John H. Ipsen

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Intrinsically Disordered Proteins ◽

Liquid Phase Separation ◽

Coarse Grained ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Wide Range ◽

End Caps ◽

Liquid Liquid Phase Separation

ABSTRACTPhase separation of immiscible fluids is a common phenomenon in polymer chemistry, and is recognized as an important mechanism by which cells compartmentalize their biochemical reactions. Biomolecular condensates are condensed fluid droplets in cells that form by liquid-liquid phase separation of intrinsically-disordered proteins. They have a wide range of functions and are associated with chronic neurodegenerative diseases in which they become pathologically rigid. Intrinsically-disordered proteins are conformationally flexible and possess multiple, distributed binding sites for each other or for RNA. However, it remains unclear how their material properties depend on the molecular structure of the proteins. Here we use coarse-grained simulations to explore the phase behavior and structure of a model biomolecular condensate composed of semi-flexible polymers with attractive end-caps in a good solvent. Although highly simplified, the model contains the minimal molecular features that are sufficient to observe liquid-liquid phase separation of soluble polymers. The polymers condense into a porous, three-dimensional network in which their end-caps reversibly bind at junctions. The spatial separation of connected junctions scales with the polymer backbone length as a self-avoiding random walk over a wide range of concentration with a weak affinity-dependent prefactor. By contrast, the average number of polymers that meet at the junctions depends strongly on the end-cap affinity but only weakly on the polymer length. The regularity and porosity of the condensed network suggests a mechanism for cells to regulate biomolecular condensates. Interaction sites along a protein may be turned on or off to modulate the condensate’s porosity and tune the diffusion and interaction of additional proteins.

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Oligonucleotides can act as superscaffolds that enhance liquid-liquid phase separation of intracellular mixtures

10.1101/2020.01.24.916858 ◽

2020 ◽

Cited By ~ 3

Author(s):

Jerelle A. Joseph ◽

Jorge R. Espinosa ◽

Ignacio Sanchez-Burgos ◽

Adiran Garaizar ◽

Daan Frenkel ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Scaffold Proteins ◽

Coarse Grained ◽

Granule Formation ◽

Coarse Grained Model ◽

Liquid Liquid Phase Separation ◽

Kinetic Barriers ◽

Oligonucleotide Binding

AbstractIntracellular liquid-liquid phase separation (LLPS) enables the formation of biomolecular condensates, which play a crucial role in the spatiotemporal organisation of biomolecules (proteins, oligonucleotides). While LLPS of biopolymers has been demonstrated in both experiments and computer simulations, the physical determinants governing phase separation of protein-oligonucleotide systems are not fully understood. Here, we introduce a minimal coarse-grained model to investigate concentration-dependent features of protein-oligonucleotide mixtures. We demonstrate that adding oligonucleotides to biomolecular condensates composed of oligonucleotide-binding scaffold proteins enhances LLPS; since oligonucleotides act as ultra-high-valency molecules (termed ‘superscaffolds’) that increase the molecular connectivity among scaffold proteins. Importantly, we find that oligonucleotides promote protein LLPS via a seeding-type mechanism; recruiting numerous protein molecules and reducing the thermodynamic and kinetic barriers for nucleation and phase separation. By probing the conformational properties of oligonucleotides within droplets, we show that these biopolymers can undergo phase separation-driven compaction, which may be entropic in nature. Finally, we provide a quantitative comparison between mixture composition, protein valency, and protein-oligonucleotide interaction strengths. We find that superscaffolds preferentially recruit higher valency proteins to condensates, and that multiphase immiscibility within condensates can be achieved by modulating the relative protein-oligonucleotide binding strengths. These results shed light on the roles of oligonucleotides in ribonu-cleoprotein granule formation, heterochromatin compaction, and internal structuring of the nucleolus and stress granules.

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Consistent Force Field Captures Homolog Resolved HP1 Phase Separation

10.1101/2021.01.06.425600 ◽

2021 ◽

Author(s):

Andrew P. Latham ◽

Bin Zhang

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Force Field ◽

Large Scale ◽

Spatial Organization ◽

Structural Difference ◽

Liquid Phase Separation ◽

Coarse Grained ◽

Disordered Proteins ◽

Liquid Liquid Phase Separation

Many proteins have been shown to function via liquid-liquid phase separation. Computational modeling could offer much needed structural details of protein condensates and reveal the set of molecular interactions that dictate their stability. However, the presence of both ordered and disordered domains in these proteins places a high demand on the model accuracy. Here, we present an algorithm to derive a coarse-grained force field, MOFF, that can model both ordered and disordered proteins with consistent accuracy. It combines maximum entropy biasing, least-squares fitting, and basic principles of energy landscape theory to ensure that MOFF recreates experimental radii of gyration while predicting the folded structures for globular proteins with lower energy. The theta temperature determined from MOFF separates ordered and disordered proteins at 300 K and exhibits a strikingly linear relationship with amino acid sequence composition. We further applied MOFF to study the phase behavior of HP1, an essential protein for posttranslational modification and spatial organization of chromatin. The force field successfully resolved the structural difference of two HP1 homologs, despite their high sequence similarity. We carried out large scale simulations with hundreds of proteins to determine the critical temperature of phase separation and uncover multivalent interactions that stabilize higher-order assemblies. In all, our work makes significant methodological strides to connect theories of ordered and disordered proteins and provides a powerful tool for studying liquid-liquid phase separation with near-atomistic details.

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Charge pattern affects the structure and dynamics of polyampholyte condensates

Physical Chemistry Chemical Physics ◽

10.1039/d0cp02764b ◽

2020 ◽

Vol 22 (34) ◽

pp. 19368-19375 ◽

Cited By ~ 3

Author(s):

Milan Kumar Hazra ◽

Yaakov Levy

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Intrinsically Disordered Proteins ◽

Liquid Phase Separation ◽

Disordered Proteins ◽

Internal Diffusion ◽

Intrinsically Disordered ◽

Structure And Dynamics ◽

Liquid Liquid Phase Separation

The charge pattern of intrinsically disordered proteins affects the dynamics and internal diffusion of their condensate formed via liquid–liquid phase separation.

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The Dynamism of Intrinsically Disordered Proteins in Liquid-Liquid Phase Separation

Biophysical Journal ◽

10.1016/j.bpj.2019.11.503 ◽

2020 ◽

Vol 118 (3) ◽

pp. 60a

Author(s):

Samrat Mukhopadhyay ◽

Anupa Majumdar ◽

Priyanka Dogra ◽

Shiny Maity ◽

Ashish Joshi

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Intrinsically Disordered Proteins ◽

Liquid Phase Separation ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

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Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid–Liquid Phase Separation

Molecules ◽

10.3390/molecules25204705 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4705

Author(s):

Adiran Garaizar ◽

Ignacio Sanchez-Burgos ◽

Rosana Collepardo-Guevara ◽

Jorge R. Espinosa

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Protein Interactions ◽

Conformational Dynamics ◽

Liquid Phase Separation ◽

Phase Behaviour ◽

Coarse Grained ◽

Protein Protein Interactions ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

Proteins containing intrinsically disordered regions (IDRs) are ubiquitous within biomolecular condensates, which are liquid-like compartments within cells formed through liquid–liquid phase separation (LLPS). The sequence of amino acids of a protein encodes its phase behaviour, not only by establishing the patterning and chemical nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviours, including conformational switching, where they transition between alternate conformational ensembles. Using Molecular Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liquid–liquid phase behaviour of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilising multivalent protein–protein interactions by reducing steric hindrance; thereby, such conformations maximize the molecular connectivity of the condensed liquid network. Extended protein configurations promote phase separation regardless of whether LLPS is driven by homotypic and/or heterotypic protein–protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liquid–liquid phase behaviour.

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Targeted modulation of protein liquid-liquid phase separation by evolution of amino-acid sequence

10.1101/2020.10.20.347542 ◽

2020 ◽

Author(s):

Simon M. Lichtinger ◽

Adiran Garaizar ◽

Rosana Collepardo-Guevara ◽

Aleks Reinhardt

Keyword(s):

Phase Separation ◽

Amino Acid ◽

Liquid Phase ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Liquid Phase Separation ◽

Computational Method ◽

Coarse Grained ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

AbstractRationally and efficiently modifying the amino-acid sequence of proteins to control their ability to undergo liquid-liquid phase separation (LLPS) on demand is not only highly desirable, but can also help to elucidate which protein features are important for LLPS. Here, we propose an innovative computational method that couples a genetic algorithm to a sequence-dependent coarse-grained protein model to evolve the amino-acid sequences of phase-separating intrinsically disordered protein regions (IDRs), and purposely enhance or inhibit their capacity to phase-separate. We apply it to the phase-separating IDRs of three naturally occurring proteins, namely FUS, hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction. We find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids. However, the direction of change in the molecular driving forces that enhance LLPS (such as hydrophobicity, aromaticity and charge) depends on the initial amino-acid sequence: the critical temperature can be enhanced by increasing the frequency of hydrophobic and aromatic residues, by changing the charge patterning, or by a combination of both. Finally, we show that the evolution of amino-acid sequences to modulate LLPS is strongly coupled to the composition of the medium (e.g. the presence or absence of RNA), which may have significant implications for our understanding of phase separation within the many-component mixtures of biological systems.

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