scholarly journals Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins

2017 ◽  
Author(s):  
Tyler S. Harmon ◽  
Alex S. Holehouse ◽  
Michael K. Rosen ◽  
Rohit V. Pappu
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Theoretical Analysis ◽  
Computer Simulations ◽  
Sol Gel ◽  
Post Translational Modifications ◽  
Intrinsically Disordered ◽  
Genetic Encoding ◽  
Interaction Domains ◽  
Reversible Phase

AbstractMany intracellular membraneless bodies appear to form via reversible phase transitions of multivalent proteins. Two relevant types of phase transitions are sol-gel transitions (gelation) and phase separation plus gelation. Gelation refers to the formation of a system spanning molecular network. This can either be enabled by phase separation or it can occur independently. Despite relevance for the formation and selectivity of compositionally distinct protein and RNA assemblies, the determinants of gelation as opposed to phase separation plus gelation remain unclear. Here, we focus on linear multivalent proteins that consist of interaction domains that are connected by disordered linkers. Using results from computer simulations and theoretical analysis we show that the lengths and sequence-specific features of disordered linkers determine the coupling between phase separation and gelation. Thus, the precise nature of phase transitions for linear multivalent proteins should be biologically tunable through genetic encoding of or post-translational modifications to linker sequences.

scholarly journals Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Tyler S Harmon ◽  
Alex S Holehouse ◽  
Michael K Rosen ◽  
Rohit V Pappu
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Physical Properties ◽  
Computer Simulations ◽  
Coarse Grained ◽  
Associative Polymers ◽  
Intrinsically Disordered ◽  
Lower Protein ◽  
Reversible Formation ◽  
Covalent Crosslinks

Phase transitions of linear multivalent proteins control the reversible formation of many intracellular membraneless bodies. Specific non-covalent crosslinks involving domains/motifs lead to system-spanning networks referred to as gels. Gelation transitions can occur with or without phase separation. In gelation driven by phase separation multivalent proteins and their ligands condense into dense droplets, and gels form within droplets. System spanning networks can also form without a condensation or demixing of proteins into droplets. Gelation driven by phase separation requires lower protein concentrations, and seems to be the biologically preferred mechanism for forming membraneless bodies. Here, we use coarse-grained computer simulations and the theory of associative polymers to uncover the physical properties of intrinsically disordered linkers that determine the extent to which gelation of linear multivalent proteins is driven by phase separation. Our findings are relevant for understanding how sequence-encoded information in disordered linkers influences phase transitions of multivalent proteins.

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

2020 ◽  
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.

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

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.

scholarly journals Ligand effects on phase separation of multivalent macromolecules

2021 ◽  
Vol 118 (10) ◽  
pp. e2017184118
Author(s):  
Kiersten M. Ruff ◽  
Furqan Dar ◽  
Rohit V. Pappu
Keyword(s):  
Phase Transitions ◽  
Phase Separation ◽  
Phase Behavior ◽  
Driving Forces ◽  
Cellular Processes ◽  
Ligand Effects ◽  
Null Effect ◽  
Cross Links ◽  
Reversible Phase

Biomolecular condensates enable spatial and temporal control over cellular processes by concentrating biomolecules into nonstoichiometric assemblies. Many condensates form via reversible phase transitions of condensate-specific multivalent macromolecules known as scaffolds. Phase transitions of scaffolds can be regulated by changing the concentrations of ligands, which are defined as nonscaffold molecules that bind to specific sites on scaffolds. Here, we use theory and computation to uncover rules that underlie ligand-mediated control over scaffold phase behavior. We use the stickers-and-spacers model wherein reversible noncovalent cross-links among stickers drive phase transitions of scaffolds, and spacers modulate the driving forces for phase transitions. We find that the modulatory effects of ligands are governed by the valence of ligands, whether they bind directly to stickers versus spacers, and the relative affinities of ligand–scaffold versus scaffold–scaffold interactions. In general, all ligands have a diluting effect on the concentration of scaffolds within condensates. Whereas monovalent ligands destabilize condensates, multivalent ligands can stabilize condensates by binding directly to spacers or destabilize condensates by binding directly to stickers. Bipartite ligands that bind to stickers and spacers can alter the structural organization of scaffold molecules within condensates even when they have a null effect on condensate stability. Our work highlights the importance of measuring dilute phase concentrations of scaffolds as a function of ligand concentration in cells. This can reveal whether ligands modulate scaffold phase behavior by enabling or suppressing phase separation at endogenous levels, thereby regulating the formation and dissolution of condensates in vivo.

Phase separation in sol gel-derived ceramic films of silica-zirconia, alumina-zirconia, and titania-zirconia system

1994 ◽  
Vol 52 ◽  
pp. 646-647
Author(s):  
J. Tong ◽  
L. Eyring
Keyword(s):  
Fracture Toughness ◽  
Phase Separation ◽  
Sol Gel ◽  
Great Influence ◽  
High Strength ◽  
Chemical Durability ◽  
Separation Method ◽  
Toughening Mechanism ◽  
Ceramic Films ◽  
Zirconia Toughened Alumina

There is increasing interest in composites containing zirconia because of their high strength, fracture toughness, and its great influence on the chemical durability in glass. For the zirconia-silica system, monolithic glasses, fibers and coatings have been obtained. There is currently a great interest in designing zirconia-toughened alumina including exploration of the processing methods and the toughening mechanism.The possibility of forming nanocrystal composites by a phase separation method has been investigated in three systems: zirconia-alumina, zirconia-silica and zirconia-titania using HREM. The morphological observations initially suggest that the formation of nanocrystal composites by a phase separation method is possible in the zirconia-alumina and zirconia-silica systems, but impossible in the zirconia-titania system. The separation-produced grain size in silica-zirconia system is around 5 nm and is more uniform than that in the alumina-zirconia system in which the sizes of the small polyhedron grains are around 10 nm. In the titania-zirconia system, there is no obvious separation as was observed in die alumina-zirconia and silica-zirconia system.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Porous Gel Coatings Obtained by Phase Separation in ORMOSIL System

2000 ◽  
Vol 628 ◽  
Author(s):  
Kazuki Nakanishi ◽  
Souichi Kumon ◽  
Kazuyuki Hirao ◽  
Hiroshi Jinnai
Keyword(s):  
Phase Separation ◽  
Reaction Time ◽  
Volume Fraction ◽  
Sol Gel ◽  
Reaction Times ◽  
Dip Coating ◽  
Coating Solution ◽  
Coating Method ◽  
Gel Phase ◽  
Dip Coating Method

ABSTRACTMacroporous silicate thick films were prepared by a sol-gel dip-coating method accompanied by the phase separation using methyl-trimethoxysilane (MTMS), nitric acid and dimethylformamide (DMF) as starting components. The morphology of the film varied to a large extent depending on the time elapsed after the hydrolysis until the dipping of the coating solution. On a glass substrate, the films prepared by early dipping had inhomogeneous submicrometer-sized pores on the surface of the film. At increased reaction times, relatively narrow sized isolated macropores were observed and their size gradually decreased with the increase of reaction time. On a polyester substrate, in contrast, micrometer-sized isolated spherical gel domains were homogeneously deposited by earlier dippings. With an increase of reaction time, the volume fraction of the gel phase increased, then the morphology of the coating transformed into co-continuous gel domains and macropores, and finally inverted into the continuous gel domains with isolated macropores. The overall morphological variation with the reaction time was explained in terms of the phase separation and the structure freezing by the forced gelation, both of which were induced by the evaporation of methanol during the dipping operation.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

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