Design of intrinsically disordered proteins that undergo phase transitions with lower critical solution temperatures

AbstractMany naturally occurring elastomers are intrinsically disordered proteins (IDPs) built up of repeating units and they can demonstrate two types of thermoresponsive phase behavior. Systems characterized by lower critical solution temperatures (LCST) undergo phase separation above the LCST whereas systems characterized by upper critical solution temperatures (UCST) undergo phase separation below the UCST. There is congruence between thermoresponsive coil-globule transitions and phase behavior whereby the theta temperatures above or below which the IDPs transition from coils to globules serve as useful proxies for the LCST / UCST values. This implies that one can design sequences with desired values for the theta temperature with either increasing or decreasing radii of gyration above the theta temperature. Here, we show that the Monte Carlo simulations performed in the so-called intrinsic solvation (IS) limit version of the temperature-dependent ABSINTH implicit solvation model, yields a useful heuristic for discriminating between sequences with known LCST versus UCST phase behavior. Accordingly, we use this heuristic in a supervised approach, integrate it with a genetic algorithm, combine this with IS limit simulations, and demonstrate that novel sequences can be designed with LCST phase behavior. These calculations are aided by direct estimates of temperature dependent free energies of solvation for model compounds that are derived using the polarizable AMOEBA forcefield. To demonstrate the validity of our designs, we calculate coil-globule transition profiles using the full ABSINTH model and combine these with Gaussian Cluster Theory calculations to establish the LCST phase behavior of designed IDPs.

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A predictive coarse-grained model for position-specific effects of post-translational modifications on disordered protein phase separation

10.1101/2020.06.12.148650 ◽

2020 ◽

Cited By ~ 4

Author(s):

T. M. Perdikari ◽

N. Jovic ◽

G. L. Dignon ◽

Y. C. Kim ◽

N. L. Fawzi ◽

...

Keyword(s):

Amino Acids ◽

Phase Separation ◽

Phase Behavior ◽

Intrinsically Disordered Proteins ◽

Coarse Grained ◽

Disordered Proteins ◽

Post Translational Modifications ◽

Coarse Grained Model ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

AbstractBiomolecules undergo liquid-liquid phase separation (LLPS) resulting in the formation of multicomponent protein-RNA membraneless organelles in cells. However, the physiological and pathological role of post translational modifications (PTMs) on the biophysics of phase behavior is only beginning to be probed. To study the effect of PTMs on LLPS in silico, we extend our transferable coarse-grained model of intrinsically disordered proteins to include phosphorylated and acetylated amino acids. Using the parameters for modified amino acids available for fixed charge atomistic forcefields, we parameterize the size and atomistic hydropathy of the coarse-grained modified amino acid beads, and hence the interactions between the modified and natural amino acids. We then elucidate how the number and position of phosphorylated and acetylated residues alter the protein’s single chain compactness and its propensity to phase separate. We show that both the number and the position of phosphorylated threonines/serines or acetylated lysines can serve as a molecular on/off switch for phase separation in the well-studied disordered regions of FUS and DDX3X, respectively. We also compare modified residues to their commonly used PTM mimics for their impact on chain properties. Importantly, we show that the model can predict and capture experimentally measured differences in the phase behavior for position-specific modifications, showing that the position of modifications can dictate phase separation. In sum, this model will be useful for studying LLPS of post-translationally modified intrinsically disordered proteins and predicting how modifications control phase behavior with position-specific resolution.Statement of SignificancePost-translational modifications are important regulators of liquid-liquid phase separation (LLPS) which drives the formation of biomolecular condensates. Theoretical methods can be used to characterize the biophysical properties of intrinsically disordered proteins (IDPs). Our recent framework for molecular simulations using a Cα-centered coarse-grained model can predict the effect of various perturbations such as mutations (Dignon et al. PloS Comput. Biol, 2018) and temperature (Dignon et al, ACS Cent. Sci., 2019) on LLPS. Here, we expand this framework to incorporate modified residues like phosphothreonine, phosphoserine and acetylysine. This model will prove useful for simulating the phase separation of post-translationally modified IDPs and predicting how position-specific modifications can control phase behavior across the large family of proteins known to be phosphorylated and acetylated.

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An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid–liquid phase separation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2100968118 ◽

2021 ◽

Vol 118 (45) ◽

pp. e2100968118

Author(s):

Aishwarya Agarwal ◽

Sandeep K. Rai ◽

Anamika Avni ◽

Samrat Mukhopadhyay

Keyword(s):

Phase Transitions ◽

Phase Separation ◽

Liquid Phase ◽

Phase Behavior ◽

Intrinsically Disordered Proteins ◽

Liquid Phase Separation ◽

Disordered Proteins ◽

Cell Physiology ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

Biomolecular condensation via liquid–liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

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Intrinsically disordered proteins access a range of hysteretic phase separation behaviors

Science Advances ◽

10.1126/sciadv.aax5177 ◽

2019 ◽

Vol 5 (10) ◽

pp. eaax5177 ◽

Cited By ~ 9

Author(s):

Felipe Garcia Quiroz ◽

Nan K. Li ◽

Stefan Roberts ◽

Patrick Weber ◽

Michael Dzuricky ◽

...

Keyword(s):

Phase Separation ◽

Phase Behavior ◽

Intrinsically Disordered Proteins ◽

Wide Spectrum ◽

Disordered Proteins ◽

Solution Temperature ◽

Intrinsically Disordered ◽

Self Assembling ◽

Phase Separation Behavior ◽

Separation Behavior

The phase separation behavior of intrinsically disordered proteins (IDPs) is thought of as analogous to that of polymers that undergo equilibrium lower or upper critical solution temperature (LCST and UCST, respectively) phase transition. This view, however, ignores possible nonequilibrium properties of protein assemblies. Here, by studying IDP polymers (IDPPs) composed of repeat motifs that encode LCST or UCST phase behavior, we discovered that IDPs can access a wide spectrum of nonequilibrium, hysteretic phase behaviors. Experimentally and through simulations, we show that hysteresis in IDPPs is tunable and that it emerges through increasingly stable interchain interactions in the insoluble phase. To explore the utility of hysteretic IDPPs, we engineer self-assembling nanostructures with tunable stability. These findings shine light on the rich phase separation behavior of IDPs and illustrate hysteresis as a design parameter to program nonequilibrium phase behavior in self-assembling materials.

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Model for disordered proteins with strongly sequence-dependent liquid phase behavior

10.1101/864942 ◽

2019 ◽

Cited By ~ 1

Author(s):

Antonia Statt ◽

Helena Casademunt ◽

Clifford P. Brangwynne ◽

Athanassios Z. Panagiotopoulos

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Phase Behavior ◽

Intrinsically Disordered Proteins ◽

Protein A ◽

Liquid Phase Separation ◽

Coarse Grained ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Liquid Liquid Phase Separation

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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Relation between single-molecule properties and phase behavior of intrinsically disordered proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804177115 ◽

2018 ◽

Vol 115 (40) ◽

pp. 9929-9934 ◽

Cited By ~ 76

Author(s):

Gregory L. Dignon ◽

Wenwei Zheng ◽

Robert B. Best ◽

Young C. Kim ◽

Jeetain Mittal

Keyword(s):

Phase Separation ◽

Phase Behavior ◽

Single Molecule ◽

Intrinsically Disordered Proteins ◽

Critical Role ◽

Coarse Grained ◽

Disordered Proteins ◽

Phase Boundaries ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

Proteins that undergo liquid–liquid phase separation (LLPS) have been shown to play a critical role in many physiological functions through formation of condensed liquid-like assemblies that function as membraneless organelles within biological systems. To understand how different proteins may contribute differently to these assemblies and their functions, it is important to understand the molecular driving forces of phase separation and characterize their phase boundaries and material properties. Experimental studies have shown that intrinsically disordered regions of these proteins are a major driving force, as many of them undergo LLPS in isolation. Previous work on polymer solution phase behavior suggests a potential correspondence between intramolecular and intermolecular interactions that can be leveraged to discover relationships between single-molecule properties and phase boundaries. Here, we take advantage of a recently developed coarse-grained framework to calculate the θ temperatureTθ, the Boyle temperatureTB, and the critical temperatureTcfor 20 diverse protein sequences, and we show that these three properties are highly correlated. We also highlight that these correlations are not specific to our model or simulation methodology by comparing between different pairwise potentials and with data from other work. We, therefore, suggest that smaller simulations or experiments to determineTθorTBcan provide useful insights into the corresponding phase behavior.

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Identifying Sequence Perturbations to an Intrinsically Disordered Protein that Determine Its Phase Separation Behavior

10.1101/2020.01.06.894576 ◽

2020 ◽

Cited By ~ 5

Author(s):

Benjamin S. Schuster ◽

Gregory L. Dignon ◽

Wai Shing Tang ◽

Fleurie M. Kelley ◽

Aishwarya Kanchi Ranganath ◽

...

Keyword(s):

Phase Separation ◽

Phase Behavior ◽

Intrinsically Disordered Proteins ◽

Lipid Membrane ◽

Coarse Grained ◽

Disordered Proteins ◽

Sequence Features ◽

Intrinsically Disordered ◽

Model Protein ◽

Arginine Residues

AbstractPhase 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 bio-inspired 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. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. 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 in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation.Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the protein’s propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

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