scholarly journals Molecular principles of recruitment and dynamics of guest proteins in liquid droplets

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kiyoto Kamagata ◽  
Nanako Iwaki ◽  
Milan Kumar Hazra ◽  
Saori Kanbayashi ◽  
Trishit Banerjee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Properties ◽  
Liquid Droplet ◽  
Disordered Proteins ◽  
Total Charge ◽  
Liquid Droplets ◽  
Host Proteins ◽  
Folded Proteins ◽  
Dynamics Simulations ◽  
Liquid Liquid Phase Separation

AbstractDespite the continuous discovery of host and guest proteins in membraneless organelles, complex host–guest interactions hinder the understanding of the molecular grammar governing liquid–liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

scholarly journals Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1756
Author(s):  
Xuchang Su ◽  
Zhi He ◽  
Lijun Meng ◽  
Hong Liang ◽  
Ruhong Zhou
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Electron Tunneling ◽  
Amyloid Β ◽  
Protein Sequencing ◽  
Disordered Proteins ◽  
Force Microscopy ◽  
Intrinsically Disordered ◽  
Atomic Force ◽  
Dynamics Simulations

Single-molecule techniques such as electron tunneling and atomic force microscopy have attracted growing interests in protein sequencing. For these methods, it is critical to refine and stabilize the protein sample to a “suitable mode” before applying a high-fidelity measurement. Here, we show that a planar heterostructure comprising boronic graphene (BC3) and nitrogenized graphene (C3N) sandwiched stripe (BC3/C3N/BC3) is capable of the effective stretching and confinement of three types of intrinsically disordered proteins (IDPs), including amyloid-β (1–42), polyglutamine (Q42), and α-Synuclein (61–95). Our molecular dynamics simulations demonstrate that the protein molecules interact more strongly with the C3N stripe than the BC3 one, which leads to their capture, elongation, and confinement along the center C3N stripe of the heterostructure. The conformational fluctuations of IDPs are substantially reduced after being stretched. This design may serve as a platform for single-molecule protein analysis with reduced thermal noise.

scholarly journals Depletion interactions modulate the binding between disordered proteins in crowded environments

2020 ◽  
Vol 117 (24) ◽  
pp. 13480-13489 ◽  
Author(s):  
Franziska Zosel ◽  
Andrea Soranno ◽  
Karin J. Buholzer ◽  
Daniel Nettels ◽  
Benjamin Schuler
Keyword(s):  
Single Molecule ◽  
Posttranslational Modifications ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Dissociation Kinetics ◽  
Cellular Targets ◽  
Intrinsically Disordered ◽  
Functional Consequences ◽  
Folded Proteins ◽  
Depletion Interactions

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.

A Short Peptide Synthon for Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Manzar Abbas ◽  
Wojciech P. Lipiński ◽  
Karina K. Nakashima ◽  
Wilhelm T.S. Huck ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Liquid Droplets ◽  
Short Peptide ◽  
Peptide Analogues ◽  
Single Peptide ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation of disordered proteins has emerged as a ubiquitous route to membraneless compartments in living cells, and similar coacervates may have played a role when the first cells formed. However, existing coacervates are typically made of multiple macromolecular components, and designing short peptide analogues capable of self-coacervation has proven difficult. Here, we present a short peptide synthon for phase separation, made of only two dipeptide stickers linked via a flexible, hydrophilic spacer. These small-molecule compounds self-coacervate into micrometre-sized liquid droplets at sub-mM concentrations, which retain up to 75 weight-% water. The design is general and we derive guidelines for the required sticker hydrophobicity and spacer polarity. To illustrate their potential as protocells, we create a disulphide-linked derivative that undergoes reversible compartmentalisation controlled by redox chemistry. The resulting coacervates sequester and melt nucleic acids, and act as microreactors that catalyse two different anabolic reactions yielding molecules of increasing complexity. This provides a stepping stone for new protocells made of single peptide species.<br>

A Short Peptide Synthon for Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
Manzar Abbas ◽  
Wojciech P. Lipiński ◽  
Karina K. Nakashima ◽  
Wilhelm T.S. Huck ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Living Cells ◽  
Liquid Phase Separation ◽  
Disordered Proteins ◽  
Liquid Droplets ◽  
Short Peptide ◽  
Peptide Analogues ◽  
Single Peptide ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation of disordered proteins has emerged as a ubiquitous route to membraneless compartments in living cells, and similar coacervates may have played a role when the first cells formed. However, existing coacervates are typically made of multiple macromolecular components, and designing short peptide analogues capable of self-coacervation has proven difficult. Here, we present a short peptide synthon for phase separation, made of only two dipeptide stickers linked via a flexible, hydrophilic spacer. These small-molecule compounds self-coacervate into micrometre-sized liquid droplets at sub-mM concentrations, which retain up to 75 weight-% water. The design is general and we derive guidelines for the required sticker hydrophobicity and spacer polarity. To illustrate their potential as protocells, we create a disulphide-linked derivative that undergoes reversible compartmentalisation controlled by redox chemistry. The resulting coacervates sequester and melt nucleic acids, and act as microreactors that catalyse two different anabolic reactions yielding molecules of increasing complexity. This provides a stepping stone for new protocells made of single peptide species.<br>

scholarly journals 1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells

2021 ◽  
Vol 4 (4) ◽  
pp. e202001005 ◽  
Author(s):  
Yuji Itoh ◽  
Shiori Iida ◽  
Sachiko Tamura ◽  
Ryosuke Nagashima ◽  
Kentaro Shiraki ◽  
...  
Keyword(s):  
Liquid Droplet ◽  
Chromatin Condensation ◽  
Human Cells ◽  
Dependent Manner ◽  
Liquid Droplets ◽  
Cellular Functions ◽  
Spatiotemporal Regulation ◽  
Dose Dependent ◽  
Liquid Liquid Phase Separation

Liquid droplets formed inside the cell by liquid–liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein–protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies. However, the effect of 1,6-HD on chromatin in living cells remains unclear. We found that 1,6-HD drastically suppresses chromatin motion and hyper-condenses chromatin in human cells by using live-cell single-nucleosome imaging, which detects changes in the state of chromatin. These effects were enhanced in a dose-dependent manner. Chromatin was “frozen” by 5%, or higher, concentrations of 1,6-HD. 1,6-HD greatly facilitated cation-dependent chromatin condensation in vitro. This 1,6-HD action is distinct from its melting activity of liquid droplets. Alcohols, such as 1,6-HD, appear to remove water molecules around chromatin and locally condense chromatin. Therefore, liquid droplet results obtained using 1,6-HD should be carefully interpreted or reconsidered when these droplets are associated with chromatin.

scholarly journals Disordered proteins follow diverse transition paths as they fold and bind to a partner

Science ◽  
2020 ◽  
Vol 368 (6496) ◽  
pp. 1253-1257 ◽  
Author(s):  
Jae-Yeol Kim ◽  
Hoi Sung Chung
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Chain Flexibility ◽  
Transition Paths ◽  
Folded Proteins

Transition paths of macromolecular conformational changes such as protein folding are predicted to be heterogeneous. However, experimental characterization of the diversity of transition paths is extremely challenging because it requires measuring more than one distance during individual transitions. In this work, we used fast three-color single-molecule Förster resonance energy transfer spectroscopy to obtain the distribution of binding transition paths of a disordered protein. About half of the transitions follow a path involving strong non-native electrostatic interactions, resulting in a transition time of 300 to 800 microseconds. The remaining half follow more diverse paths characterized by weaker electrostatic interactions and more than 10 times shorter transition path times. The chain flexibility and non-native interactions make diverse binding pathways possible, allowing disordered proteins to bind faster than folded proteins.

scholarly journals Solvent-dependent segmental dynamics in intrinsically disordered proteins

2019 ◽  
Vol 5 (6) ◽  
pp. eaax2348 ◽  
Author(s):  
Nicola Salvi ◽  
Anton Abyzov ◽  
Martin Blackledge
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Dynamic Properties ◽  
Nmr Relaxation ◽  
Bulk Water ◽  
Water Dynamics ◽  
Disordered Proteins ◽  
Segmental Dynamics ◽  
Intrinsically Disordered ◽  
Dynamics Simulations ◽  
And Function

Protein and water dynamics have a synergistic relationship, which is particularly important for intrinsically disordered proteins (IDPs), although the details of this coupling remain poorly understood. Here, we combine temperature-dependent molecular dynamics simulations using different water models with extensive nuclear magnetic resonance (NMR) relaxation to examine the importance of distinct modes of solvent and solute motion for the accurate reproduction of site-specific dynamics in IDPs. We find that water dynamics play a key role in motional processes internal to “segments” of IDPs, stretches of primary sequence that share dynamic properties and behave as discrete dynamic units. We identify a relationship between the time scales of intrasegment dynamics and the lifetime of hydrogen bonds in bulk water. Correct description of these motions is essential for accurate reproduction of protein relaxation. Our findings open important perspectives for understanding the role of hydration water on the behavior and function of IDPs in solution.

scholarly journals Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

2020 ◽  
Author(s):  
Tomoto Ura ◽  
Ako Kagawa ◽  
Hiromasa Yagi ◽  
Naoya Tochio ◽  
Takanori Kigawa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Living Cells ◽  
Enzyme Activation ◽  
Liquid Phase Separation ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Essential Information ◽  
Liquid Liquid Phase Separation

ABSTRACTLiquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

scholarly journals Spatiotemporal solidification of α-synuclein inside the liquid droplets

2021 ◽  
Author(s):  
Soumik Ray ◽  
Debdeep Chatterjee ◽  
Semanti Mukherjee ◽  
Komal Patel ◽  
Jaladhar K Mahato ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Protein Aggregation ◽  
Neurodegenerative Disorders ◽  
Physical Mechanism ◽  
Liquid Droplet ◽  
Liquid Droplets ◽  
Single Droplet ◽  
Liquid To Solid Transition ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) and subsequent liquid-to-solid transition is implicated in membraneless organelles formation as well as disease associated protein aggregation. However, how liquid-to-solid transition is initiated inside a liquid droplet remains unclear. Here, using studies at single droplet resolution, we show that liquid-to-solid transition of α-synuclein (α-Syn) liquid droplets is associated with significant changes in the local microenvironment as well as secondary structure of the protein, which is prominently observed at the center of the liquid droplets. With the ageing of liquid droplets, the structured core at the center gradually expands and propagates over entire droplets. Further, during droplet fusion, smaller, homogeneous droplets progressively dissolve and supply proteins to the larger, heterogeneous droplets containing solid-like core at their center. The present study will significantly help to under-stand the physical mechanism of LLPS and liquid-to-solid transition in biological compartmentalization as well as in protein aggregation associated with human neurodegenerative disorders.

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