scholarly journals Supramolecular Fuzziness of Intracellular Liquid Droplets: Liquid–Liquid Phase Transitions, Membrane-Less Organelles, and Intrinsic Disorder

Molecules ◽  
2019 ◽  
Vol 24 (18) ◽  
pp. 3265 ◽  
Author(s):  
Vladimir N. Uversky
Keyword(s):  
Phase Transitions ◽  
Liquid Phase ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Disordered Proteins ◽  
Bacterial Cells ◽  
Liquid Droplets ◽  
Intrinsically Disordered ◽  
Wide Range ◽  
Characteristic Features

Cells are inhomogeneously crowded, possessing a wide range of intracellular liquid droplets abundantly present in the cytoplasm of eukaryotic and bacterial cells, in the mitochondrial matrix and nucleoplasm of eukaryotes, and in the chloroplast’s stroma of plant cells. These proteinaceous membrane-less organelles (PMLOs) not only represent a natural method of intracellular compartmentalization, which is crucial for successful execution of various biological functions, but also serve as important means for the processing of local information and rapid response to the fluctuations in environmental conditions. Since PMLOs, being complex macromolecular assemblages, possess many characteristic features of liquids, they represent highly dynamic (or fuzzy) protein–protein and/or protein–nucleic acid complexes. The biogenesis of PMLOs is controlled by specific intrinsically disordered proteins (IDPs) and hybrid proteins with ordered domains and intrinsically disordered protein regions (IDPRs), which, due to their highly dynamic structures and ability to facilitate multivalent interactions, serve as indispensable drivers of the biological liquid–liquid phase transitions (LLPTs) giving rise to PMLOs. In this article, the importance of the disorder-based supramolecular fuzziness for LLPTs and PMLO biogenesis is discussed.

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 Liquid-Liquid Phase Separation is Driven by Large-Scale Conformational Unwinding and Fluctuations of Intrinsically Disordered Protein Molecules

2019 ◽  
Author(s):  
Anupa Majumdar ◽  
Priyanka Dogra ◽  
Shiny Maity ◽  
Samrat Mukhopadhyay
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Large Scale ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Fluorescence Depolarization ◽  
Intrinsically Disordered ◽  
Liquid Liquid Phase Separation

ABSTRACTLiquid-liquid phase separation occurs via a multitude of transient, non-covalent, intermolecular interactions resulting in phase transition of intrinsically disordered proteins/regions (IDPs/IDRs) and other biopolymers into mesoscopic, dynamic, non-stoichiometric, supramolecular condensates. IDPs resemble associative polymers possessing stereospecific “stickers” and flexible “spacers” that govern the transient chain-chain interactions and fluidity in phase-separated liquid droplets. However, the fundamental molecular origin of phase separation remains elusive. Here we present a unique case to demonstrate that unusual conformational expansion events coupled with solvation and fluctuations drive phase separation of tau, an IDP associated with Alzheimer’s disease. Using intramolecular excimer emission as a powerful proximity readout, we show the unraveling of polypeptide chains within the protein-rich interior environment that can promote critical interchain contacts. Using highly-sensitive picosecond time-resolved fluorescence depolarization measurements, we directly capture rapid large-amplitude torsional fluctuations in the extended chains that can control the relay of making-and-breaking of noncovalent intermolecular contacts maintaining the internal fluidity. Our observations, together with the existing polymer theories, suggest that such an orchestra of concerted molecular shapeshifting events involving chain expansion, solvation, and fluctuations can provide additional favorable free energies to overcome the entropy of mixing term during phase separation. The interplay of these key molecular parameters can also be of prime importance in modulating the mesoscale material property of liquid-like condensates and their maturation of into pathological gel-like and solid-like aggregates.

scholarly journals Structure of biomolecular condensates from dissipative particle dynamics simulations

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.

scholarly journals Generation of the configurational ensemble of an intrinsically disordered protein from unbiased molecular dynamics simulation

2019 ◽  
Vol 116 (41) ◽  
pp. 20446-20452 ◽  
Author(s):  
Utsab R. Shrestha ◽  
Puneet Juneja ◽  
Qiu Zhang ◽  
Viswanathan Gurumoorthy ◽  
Jose M. Borreguero ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Intrinsically Disordered Proteins ◽  
Chemical Shifts ◽  
Intrinsically Disordered Protein ◽  
Physical Models ◽  
Dynamics Simulation ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Eukaryotic Proteomes ◽  
Heterogeneous Ensemble

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes, play a major role in cell signaling, and are associated with human diseases. To understand IDP function it is critical to determine their configurational ensemble, i.e., the collection of 3-dimensional structures they adopt, and this remains an immense challenge in structural biology. Attempts to determine this ensemble computationally have been hitherto hampered by the necessity of reweighting molecular dynamics (MD) results or biasing simulation in order to match ensemble-averaged experimental observables, operations that reduce the precision of the generated model because different structural ensembles may yield the same experimental observable. Here, by employing enhanced sampling MD we reproduce the experimental small-angle neutron and X-ray scattering profiles and the NMR chemical shifts of the disordered N terminal (SH4UD) of c-Src kinase without reweighting or constraining the simulations. The unbiased simulation results reveal a weakly funneled and rugged free energy landscape of SH4UD, which gives rise to a heterogeneous ensemble of structures that cannot be described by simple polymer theory. SH4UD adopts transient helices, which are found away from known phosphorylation sites and could play a key role in the stabilization of structural regions necessary for phosphorylation. Our findings indicate that adequately sampled molecular simulations can be performed to provide accurate physical models of flexible biosystems, thus rationalizing their biological function.

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

APL Materials ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 021119 ◽  
Author(s):  
Xiangze Zeng ◽  
Chengwen Liu ◽  
Martin J. Fossat ◽  
Pengyu Ren ◽  
Ashutosh Chilkoti ◽  
...  
Keyword(s):  
Phase Transitions ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Critical Solution Temperatures

scholarly journals Enhancing Binding Affinity of an Intrinsically Disordered Protein by α-Methylation of Key Amino Acid Residues

2019 ◽  
Author(s):  
Valentin Bauer ◽  
Boris Schmidtgall ◽  
Gergő Gógl ◽  
Jozica Dolenc ◽  
Judit Osz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Selective Inhibition ◽  
Disordered Proteins ◽  
Creb Binding Protein ◽  
Alpha Helices ◽  
Intrinsically Disordered ◽  
Order Of Magnitude

Intrinsically disordered proteins (IDPs), which undergo folding upon binding to their targets, are critical players in protein interaction networks. Here we demonstrate that incorporation of non-canonical alpha-methylated amino acids into the unstructured activation domain of the transcriptional coactivator ACTR can stabilize helical conformations and strengthen binding interactions with the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP). A combinatorial alpha-methylation scan of the ACTR sequence converged on two substitutions at positions 1055 and 1076 that increase affinity for both NCBD and the full length 270 kDa CBP by one order of magnitude. The first X-ray structure of the modified ACTR domain bound to NCBD revealed that the key alpha-methylated amino acids were localized within alpha-helices. Biophysical studies showed that the observed changes in binding energy are the result of long-range interactions and redistribution of enthalpy and entropy. This proof-of-concept study establishes a potential strategy for selective inhibition of protein-protein interactions involving IDPs in cells.<br>

An intrinsically disordered protein, CP12: jack of all trades and master of the Calvin cycle

2012 ◽  
Vol 40 (5) ◽  
pp. 995-999 ◽  
Author(s):  
Brigitte Gontero ◽  
Stephen C. Maberly
Keyword(s):  
Amino Acids ◽  
Stress Responses ◽  
Intrinsically Disordered Proteins ◽  
Calvin Cycle ◽  
Intrinsically Disordered Protein ◽  
Disulfide Bridge ◽  
Co2 Assimilation ◽  
Disordered Proteins ◽  
Dimensional Structure ◽  
Intrinsically Disordered

Many proteins contain disordered regions under physiological conditions and lack specific three-dimensional structure. These are referred to as IDPs (intrinsically disordered proteins). CP12 is a chloroplast protein of approximately 80 amino acids and has a molecular mass of approximately 8.2–8.5 kDa. It is enriched in charged amino acids and has a small number of hydrophobic residues. It has a high proportion of disorder-promoting residues, but has at least two (often four) cysteine residues forming one (or two) disulfide bridge(s) under oxidizing conditions that confers some order. However, CP12 behaves like an IDP. It appears to be universally distributed in oxygenic photosynthetic organisms and has recently been detected in a cyanophage. The best studied role of CP12 is its regulation of the Calvin cycle responsible for CO2 assimilation. Oxidized CP12 forms a supramolecular complex with two key Calvin cycle enzymes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and PRK (phosphoribulokinase), down-regulating their activity. Association–dissociation of this complex, induced by the redox state of CP12, allows the Calvin cycle to be inactive in the dark and active in the light. CP12 is promiscuous and interacts with other enzymes such as aldolase and malate dehydrogenase. It also plays other roles in plant metabolism such as protecting GAPDH from inactivation and scavenging metal ions such as copper and nickel, and it is also linked to stress responses. Thus CP12 seems to be involved in many functions in photosynthetic cells and behaves like a jack of all trades as well as being a master of the Calvin cycle.

Charge pattern affects the structure and dynamics of polyampholyte condensates

2020 ◽  
Vol 22 (34) ◽  
pp. 19368-19375 ◽  
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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