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Free energy calculations on solid phases are important for understanding the phase behavior of various systems. For periodic crystalline solids, the Einstein molecule method can be used to determine the free energy difference between the solid of interest and an ideal crystal for which the free energy can be found analytically. In this work, we show how this method is extensible to systems which are nonperiodic or periodic in some dimensions but not in others. This allows for the calculation of exact absolute free energies of finite-sized crystals having specific shapes and surface geometries. We demonstrate this using the fcc Lennard-Jones solid and also illustrate how surface contributions to free energies can easily be extracted from simulations of this solid in semi-infinite slab geometries. We have developed a software package which interfaces with the LAMMPS molecular dynamics code to perform these calculations. [ABSTRACT FROM AUTHOR]

Coarse-grained (CG) protein models in the structural biology literature have improved over the years from being simple tools to understand general folding and aggregation driving forces to capturing detailed structures achieved by actual folding sequences. Here, we ask whether such models can be developed systematically from recent advances in bottom-up coarse-graining methods without relying on bioinformatic data (e.g., protein data bank statistics). We use relative entropy coarse-graining to develop a hybrid CG but G o ¯ -like CG peptide model, hypothesizing that the landscape of proteinlike folds is encoded by the backbone interactions, while the sidechain interactions define which of these structures globally minimizes the free energy in a unique native fold. To construct a model capable of capturing varied secondary structures, we use a new extended ensemble relative entropy method to coarse-grain based on multiple reference atomistic simulations of short polypeptides with varied α and β character. Subsequently, we assess the CG model as a putative protein backbone forcefield by combining it with sidechain interactions based on native contacts but not incorporating native distances explicitly, unlike standard G o ¯ models. We test the model's ability to fold a range of proteins and find that it achieves high accuracy (∼2 Å root mean square deviation resolution for both short sequences and large globular proteins), suggesting the strong role that backbone conformational preferences play in defining the fold landscape. This model can be systematically extended to non-natural amino acids and nonprotein polymers and sets the stage for extensions to non-G o ¯ models with sequence-specific sidechain interactions. [ABSTRACT FROM AUTHOR]

The fluid phase diagram of trimer particles composed of one central attractive bead and two repulsive beads was determined as a function of simple geometric parameters using flat-histogram Monte Carlo methods. A variety of self-assembled structures were obtained including spherical micelle-like clusters, elongated clusters, and densely packed cylinders, depending on both the state conditions and shape of the trimer. Advanced simulation techniques were employed to determine transitions between self-assembled structures and macroscopic phases using thermodynamic and structural definitions. Simple changes in particle geometry yield dramatic changes in phase behavior, ranging from macroscopic fluid phase separation to molecular-scale self-assembly. In special cases, both self-assembled, elongated clusters and bulk fluid phase separation occur simultaneously. Our work suggests that tuning particle shape and interactions can yield superstructures with controlled architecture. [ABSTRACT FROM AUTHOR]

DNA-functionalized particles have great potential for the design of complex self-assembled materials. The major hurdle in realizing crystal structures from DNA-functionalized particles is expected to be kinetic barriers that trap the system in metastable amorphous states. Therefore, it is vital to explore the molecular details of particle assembly processes in order to understand the underlying mechanisms. Molecular simulations based on coarse-grained models can provide a convenient route to explore these details. Most of the currently available coarse-grained models of DNA-functionalized particles ignore key chemical and structural details of DNA behavior. These models therefore are limited in scope for studying experimental phenomena. In this paper, we present a new coarsegrained model of DNA-functionalized particles which incorporates some of the desired features of DNA behavior. The coarse-grained DNA model used here provides explicit DNA representation (at the nucleotide level) and complementary interactions between Watson-Crick base pairs, which lead to the formation of single-stranded hairpin and double-stranded DNA. Aggregation between multiple complementary strands is also prevented in our model. We study interactions between two DNA-functionalized particles as a function of DNA grafting density, lengths of the hybridizing and non-hybridizing parts of DNA, and temperature. The calculated free energies as a function of pair distance between particles qualitatively resemble experimental measurements of DNA-mediated pair interactions. [ABSTRACT FROM AUTHOR]

We use discontinuous molecular dynamics and grand-canonical transition-matrix Monte Carlo simulations to explore how confinement between parallel hard walls modifies the relationships between packing fraction, self-diffusivity, partial molar excess entropy, and total excess entropy for binary hard-sphere mixtures. To accomplish this, we introduce an efficient algorithm to calculate partial molar excess entropies from the transition-matrix Monte Carlo simulation data. We find that the species-dependent self-diffusivities of confined fluids are very similar to those of the bulk mixture if compared at the same, appropriately defined, packing fraction up to intermediate values, but then deviate negatively from the bulk behavior at higher packing fractions. On the other hand, the relationships between self-diffusivity and partial molar excess entropy (or total excess entropy) observed in the bulk fluid are preserved under confinement even at relatively high packing fractions and for different mixture compositions. This suggests that the excess entropy, calculable from classical density functional theories of inhomogeneous fluids, can be used to predict some of the nontrivial dynamical behaviors of fluid mixtures in confined environments. [ABSTRACT FROM AUTHOR]

We present molecular simulation data for viscosity, self-diffusivity, and the local structural ordering of (i) a hard-sphere fluid and (ii) a square-well fluid with short-range attractions. The latter fluid exhibits a region of dynamic anomalies in its phase diagram, where its mobility increases upon isochoric cooling, which is found to be a subset of a larger region of structural anomalies, in which its pair correlations strengthen upon isochoric heating. This “cascade of anomalies” qualitatively resembles that found in recent simulations of liquid water. The results for the hard-sphere and square-well systems also show that the breakdown of the Stokes–Einstein relation upon supercooling occurs for conditions where viscosity and self-diffusivity develop different couplings to the degree of pairwise structural ordering of the liquid. We discuss how these couplings reflect dynamic heterogeneities. Finally, we note that the simulation data suggest how repulsive and attractive glasses may generally be characterized by two distinct levels of short-range structural order. [ABSTRACT FROM AUTHOR]

We use grand canonical transition-matrix Monte Carlo and discontinuous molecular dynamics simulations to generate precise thermodynamic and kinetic data for the equilibrium hard-sphere fluid confined between smooth hard walls. These simulations show that the pronounced inhomogeneous structuring of the fluid normal to the confining walls, often the primary focus of density functional theory studies, has a negligible effect on many of its average properties over a surprisingly broad range of conditions. We present one consequence of this insensitivity to confinement: a simple analytical equation relating the average density of the confined fluid to that of the bulk fluid with equal activity. Nontrivial implications of confinement for average fluid properties do emerge in this system, but only when the fluid is both (i) dense and (ii) confined to a gap smaller than approximately three particle diameters. For this limited set of conditions, we find that “in-phase” oscillatory deviations in excess entropy and self-diffusivity (relative to the behavior of the bulk fluid at the same average density) occur as a function of gap size. These paired thermodynamic/kinetic deviations from bulk behavior appear to reflect the geometric packing frustration that arises when the confined space cannot naturally accommodate an integer number of particle layers. [ABSTRACT FROM AUTHOR]

Glycosylation is one of the most common post-translational modifications (PTMs), which provides a large proteome diversity. Previous work on glycosylation of globular proteins has revealed remarkable effects of glycosylation on protein function, altering the folding stability and structure and/or altering the protein surface which affects their binding characteristics. Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of large proteins are also frequently glycosylated, yet how glycosylation affects their function remains to be elucidated. An important open question is, does glycosylation affect IDP structure or binding characteristics or both? In this work, we particularly address the structural effects of O-linked glycosylation by investigating glycosylated and unglycosylated forms of two different IDPs, tau174-183 and human islet amyloid polypeptide (hIAPP), by all-atom explicit solvent simulations. We simulate these IDPs in aqueous solution for O-linked glycosylated and unglycosylated forms by employing two modern all-atom force fields for which glycan parameters are also available. We find that O-linked glycosylation only has a modest effect on equilibrium structural ensembles of IDPs, for the cases studied here, which suggests that the functional role of glycosylation may be primarily exerted by modulation of the protein binding characteristics rather than structure. [ABSTRACT FROM AUTHOR]

We used single molecule force spectroscopy to measure the force required to remove single-stranded DNA (ssDNA) homopolymers from single-walled carbon nanotubes (SWCNTs) deposited on methyl-terminated self-assembled monolayers (SAMs). The peeling forces obtained from these experiments are bimodal in distribution. The cluster of low forces corresponds to peeling from the SAM surface, while the cluster of high forces corresponds to peeling from the SWCNTs. Using a simple equilibrium model of the single molecule peeling process, we calculated the free energy of binding per nucleotide. We found that the free energy of ssDNA binding to hydrophobic SAMs decreases as poly(A) > poly(G) ≈ poly(T) > poly(C) (16.9 ± 0.1; 9.7 ± 0.1; 9.5 ± 0.1; 8.7 ± 0.1 kBT, per nucleotide). The free energy of ssDNA binding to SWCNT adsorbed on this SAM also decreases in the same order poly(A) > poly(G) > poly(T) > poly(C), but its magnitude is significantly greater than that of DNA—SAM binding energy (38.1 ± 0.2; 33.9 ± 0.1; 23.3 ± 0.1; 17.1 ± 0.1 kBT, per nucleotide). An unexpected finding is that binding strength of ssDNA to the curved SWCNTs is much greater than to flat graphite, which also has a different ranking (poly(T) > poly(A) > poly(G) > poly(C); 11.3 ± 0.8, 9.9 ± 0.5, 8.3 ± 0.2, and 7.5 ± 0.8 kBT, respectively, per nucleotide). Replica-exchange molecular dynamics simulations show that ssDNA binds preferentially to the curved SWCNT surface, leading us to conclude that the differences in ssDNA binding between graphite and nanotubes arise from the spontaneous curvature of ssDNA. [ABSTRACT FROM AUTHOR]

A statistical mechanical theory is presented to predict the effects of macromolecular crowding on protein association equilibria, accounting for both excluded volume and attractive interactions between proteins and crowding molecules. Predicted binding free energies are in excellent agreement with simulation data over a wide range of crowder sizes and packing fractions. It is shown that attractive interactions between proteins and crowding agents counteract the stabilizing effects of excluded volume interactions. A critical attraction strength, for which there is no net effect of crowding, is approximately independent of the crowder packing fraction. [ABSTRACT FROM AUTHOR]

Intrinsically disordered proteins (IDPs), which playkey rolesin cell signaling and regulation, do not display specific tertiarystructure when isolated in solution. Instead, they dynamically explorean ensemble of unfolded configurations, adopting more stable, orderedstructures only after binding to their ligands. Whether ligands induceIDP structural changes upon binding or simply bind to pre-existingconformers that are populated within the IDP’s structural ensembleis not well understood. Molecular simulations can provide informationwith the spatiotemporal resolution necessary to resolve these issues.Here, we report on the conformational ensemble of a 15-residue wild-typep53 fragment from the TAD domain and its mutant (TAD-P27L) obtainedby replica exchange molecular dynamics simulation using an optimized(fully atomistic, explicit solvent) protein model and the experimentalvalidation of the simulation results. We use a clustering method basedon structural similarity to identify conformer states populated bythe peptides in solution from the simulated ensemble. We show thatp53 populates solution structures that strongly resemble the ligand(MDM2)-bound structure, but at the same time, the conformational free-energylandscape is relatively flat in the absence of the ligand. [ABSTRACT FROM AUTHOR]

We have performed the first unbiased folding simulations of the GB1 hairpin in explicit solvent, using hundreds of microsecond-long molecular dynamics simulations (total time: 0.7 ms). Our simulations are initiated from two sets of structures. Starting from an equilibrium unfolded state, we obtain single-exponential folding kinetics with rate coefficients in good agreement (T = 350 K) or within an order of magnitude (T = 300 K) of the experimental values. However, simulations initiated from unfolded configurations lacking secondary structure result in biexponential kinetics with an additional fast nanosecond kinetic mode. This mode can strongly bias the folding rate estimated from the mean first passage time, when the trials are much shorter than the folding time. We find that the mechanism of the hairpin folding is insensitive to the details of the initial unfolded ensemble and is initiated by correct formation of the turn of the hairpin, followed by the formation of the native hydrogen bonds and hydrophobic contacts, consistent with experimental Φ-value analysis. Subsequent native interactions can be formed either from the turn or from the hairpin termini, helping to explain an apparent discrepancy in experimental results. From our simulations, we also obtain the transition path durations, a critical parameter for single molecule experiments aiming to resolve events along folding pathways. The lengths of transition paths span a wide range, from 50 ps to 140 ns, at 300 K. [ABSTRACT FROM AUTHOR]

Understanding the effects of confinement on protein stability and folding kinetics is important for describing protein folding in the cellular environment. We have investigated the effects of confinement on two structurally distinct proteins as a function of the dimension dc and characteristic size R of the confining boundary. We find that the stabilization of the folded state relative to bulk conditions is quantitatively described by R-γc, where the exponent γc is ≈5/3 independent of the dimension of confinement dc (cylindrical, planar, or spherical). Moreover, we find that the logarithm of the folding rates also scale as R-γc, with deviations only being seen for very small confining geometries, where folding is downhill; for both stability and kinetics, the dominant effect is the change in the free energy of the unfolded state. A secondary effect on the kinetics is a slight destabilization of the transition state by confinement, although the contacts present in the confined transition state are essentially identical to the bulk case. We investigate the effect of confinement on the position-dependent diffusion coefficients D(Q) for dynamics along the reaction coordinate Q (fraction of native contacts). The diffusion coefficients only change in the unfolded state basin, where they are increased because of compaction. [ABSTRACT FROM AUTHOR]

We study the static and dynamic properties of the water-density fluctuations in the interface of large nonpolar solutes. With the help of extensive molecular dynamics simulations of TIP4P water near smooth spherical solutes, we show that for large solutes, the interfacial density profile is broadened by capillary waves. For purely repulsive solutes, the squared width of the interface increases linearly with the logarithm of the solute size, as predicted by capillary-wave theory. The apparent interfacial tension extracted from the slope agrees with that of a free liquid-vapor interface. The characteristic length of local density fluctuations is ≈0.5 nm, measured along the arc, again consistent with that of a free liquid-vapor interface. Probed locally, the interfacial density fluctuations exhibit large variances that exceed those expected for an ideal gas. Qualitatively consistent with theories of the free liquid-vapor interface, we find that the water interface near large and strongly nonpolar solutes is flickering, broadened by capillary-wave fluctuations. These fluctuations result in transitions between locally wet and dry regions that are slow on a molecular time scale. [ABSTRACT FROM AUTHOR]

Diffusivity, a measure for how rapidly a fluid self-mixes, shows an intimate, but seemingly fragmented, connection to thermodynamics. On one hand, the “configurational” contribution to entropy (related to the number of mechanically stable configurations that fluid molecules can adopt) has long been considered key for predicting supercooled liquid dynamics near the glass transition. On the other hand, the excess entropy (relative to ideal gas) provides a robust scaling for the diffusivity of fluids above the freezing point. Here we provide, to our knowledge, the first evidence that excess entropy also captures how supercooling a fluid modifies its diffusivity, suggesting that dynamics, from ideal gas to glass, is related to a single, standard thermodynamic quantity. [ABSTRACT FROM AUTHOR]

We calculate the pair diffusion coefficient D(r) as a function of the distance r between two hard sphere particles in a dense monodisperse fluid. The distance-dependent pair diffusion coefficient describes the hydrodynamic interactions between particles in a fluid that are central to theories of polymer and colloid dynamics. We determine D(r) from the propagators (Green's functions) of particle pairs obtained from molecular dynamics simulations. At distances exceeding ∼3 molecular diameters, the calculated pair diffusion coefficients are in excellent agreement with predictions from exact macroscopic hydrodynamic theory for large Brownian particles suspended in a solvent bath, as well as the Oseen approximation. However, the asymptotic 1/r distance dependence of D(r) associated with hydrodynamic effects emerges only after the pair distance dynamics has been followed for relatively long times, indicating non-negligible memory effects in the pair diffusion at short times. Deviations of the calculated D(r) from the hydrodynamic models at short distances r reflect the underlying many-body fluid structure, and are found to be correlated to differences in the local available volume. The procedure used here to determine the pair diffusion coefficients can also be used for single-particle diffusion in confinement with spherical symmetry. [ABSTRACT FROM AUTHOR]

A correction to the article "Relationship between thermodynamics and dynamics of supercooled liquids" that was published in the April 28, 2010 issue is presented.

The heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1α, HP1β, and HP1γ, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1α with HP1β and HP1γ versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization. [ABSTRACT FROM AUTHOR]

Biomolecular phase separation that contributes to the formation of membraneless organelles and biomolecular condensates has recently gained tremendous attention because of the importance of these assemblies in physiology, disease, and engineering applications. Understanding and directing biomolecular phase separation requires a multiscale view of the biophysical properties of these phases. Yet, many classic tools to characterize biomolecular properties do not apply in these condensed phases. Here, we discuss insights obtained from spectroscopic methods, in particular nuclear magnetic resonance and optical spectroscopy, in understanding the molecular and atomic interactions that underlie the formation of protein-rich condensates. We also review approaches closely coupling nuclear magnetic resonance data with computational methods especially coarse-grained and all-atom molecular simulations, which provide insight into molecular features of phase separation. Finally, we point to future methodolical developments, particularly visualizing biophysical properties of condensates in cells. [ABSTRACT FROM AUTHOR]

Molecular dynamics simulations are used to study the occupancy and flow of water through nanotubes comprised of hydrophobic and hydrophilic atoms, which are arranged on a honeycomb lattice to mimic functionalized carbon nanotubes (CNTs). We consider single-file motion of TIP3P water through narrow channels of (6,6) CNTs with varying fractions (f) of hydrophilic atoms. Various arrangements of hydrophilic atoms are used to create heterogeneous nanotubes with separate hydrophobic/hydrophilic domains along the tube as well as random mixtures of the two types of atoms. The water occupancy inside the nanotube channel is found to vary nonlinearly as a function of f, and a small fraction of hydrophilic atoms (f ≈ 0.4) are sufficient to induce spontaneous and continuous filling of the nanotube. Interestingly, the average number of water molecules inside the channel and water flux through the nanotube are less sensitive to the specific arrangement of hydrophilic atoms than to the fraction, f, Two different regimes are observed for the water flux dependence on f - an approximately linear increase in flux as a function of f for f < 0.4, and almost no change in flux for higher f values, similar to the change in water occupancy. We are able to define an effective interaction strength between nanotube atoms and water's oxygen, based on a linear combination of interaction strengths between hydrophobic and hydrophilic nanotube atoms and water, that can quantitatively capture the observed behavior. [ABSTRACT FROM AUTHOR]

• Liquid-liquid phase separation underlies the formation of membraneless organelles within cells. • Theory and molecular simulations help to relate interactions at the molecular level to macroscopic properties of biomolecular condensates. • Atomistic simulation models are helpful to predict local structural motifs and atomic interaction modes. • Coarse-grained simulation models and theory can be used to predict phase equilibria from sequence properties. • Polymer field theory models can help bridge the gap between molecular simulations of single IDPs and a complete description of phase separation. Intrinsically disordered proteins (IDPs) are an important class of proteins that do not fold to a well-defined three-dimensional shape but rather adopt an ensemble of inter-converting conformations. This feature makes their experimental characterization challenging and invites a theoretical and computational approach to complement experimental studies. In this review, we highlight the recent progress in developing new computational and theoretical approaches to study the structure and dynamics of monomeric and order higher assemblies of IDPs, with a particular emphasis on their phase separation into protein-rich condensates. [ABSTRACT FROM AUTHOR]

Short-chain dehydrogenases/reductases (SDRs) are one of the most prevalent enzyme families distributed among the sequenced microorganisms. Despite the presence of a conserved catalytic tetrad and high structural similarity, these enzymes exhibit different substrate specificities. The insufficient knowledge regarding the amino acids underlying substrate specificity hinders the understanding of the SDRs' roles in diverse and significant biological processes. Here, we performed bioinformatic analysis, molecular modeling, and mutagenesis studies to identify the key residues that regulate the substrate specificities of two homologous microbial SDRs (i.e., DesE and KduD). Further, we investigated the impact of altering the physicochemical properties of these amino acids on enzyme activity. Interestingly, molecular dynamics simulations also suggest a critical role of enzyme conformational flexibility in substrate recognition and catalysis. Overall, our findings improve the understanding of microbial SDR substrate specificity and shed light on future rational design of more efficient and effective biocatalysts. [ABSTRACT FROM AUTHOR]

Self-assembly of isotropically interacting particles into desired crystal structures could allow for creating designed functional materials via simple synthetic means. However, the ability to use isotropic particles to assemble different crystal types remains challenging, especially for generating low-coordinated crystal structures. Here, we demonstrate that isotropic pairwise interparticle interactions can be rationally tuned through the design of DNA shells in a range that allows transition from common, high-coordinated FCC-CuAu and BCC-CsCl lattices, to more exotic symmetries for spherical particles such as the SC-NaCl lattice and to low-coordinated crystal structures (i.e., cubic diamond, open honeycomb). The combination of computational and experimental approaches reveals such a design strategy using DNA-functionalized nanoparticles and successfully demonstrates the realization of BCC-CsCl, SC-NaCl, and a weakly ordered cubic diamond phase. The study reveals the phase behavior of isotropic nanoparticles for DNA-shell tunable interaction, which, due to the ease of synthesis is promising for the practical realization of non-close-packed lattices. [ABSTRACT FROM AUTHOR]

Biological phase separation is known to be important for cellular organization, which has recently been extended to a new class of biomolecules that form liquid-like droplets coexisting with the surrounding cellular or extracellular environment. These droplets are termed membraneless organelles, as they lack a dividing lipid membrane, and are formed through liquid-liquid phase separation (LLPS). Elucidating the molecular determinants of phase separation is a critical challenge for the field, as we are still at the early stages of understanding how cells may promote and regulate functions that are driven by LLPS. In this review, we discuss the role that disorder, perturbations to molecular interactions resulting from sequence, posttranslational modifications, and various regulatory stimuli play on protein LLPS, with a particular focus on insights that may be obtained from simulation and theory. We finally discuss how these molecular driving forces alter multicomponent phase separation and selectivity. [ABSTRACT FROM AUTHOR]

Biological phase separation is known to be important for cellular organization, which has recently been extended to a new class of biomolecules that form liquid-like droplets coexisting with the surrounding cellular or extracellular environment. These droplets are termed membraneless organelles, as they lack a dividing lipid membrane, and are formed through liquid-liquid phase separation (LLPS). Elucidating the molecular determinants of phase separation is a critical challenge for the field, as we are still at the early stages of understanding how cells may promote and regulate functions that are driven by LLPS. In this review, we discuss the role that disorder, perturbations to molecular interactions resulting from sequence, posttranslational modifications, and various regulatory stimuli play on protein LLPS, with a particular focus on insights that may be obtained from simulation and theory. We finally discuss how these molecular driving forces alter multicomponent phase separation and selectivity. [ABSTRACT FROM AUTHOR]

Functionalisation of colloidal particles with DNA provides a powerful and flexible path towards self-assembly of ordered materials. Given the nearly limitless possibilities for constructing DNA-functionalised particles, and the wide range of conditions under which they can be assembled, it is crucial to gain an understanding of the principles governing self-assembly of these particles and how their properties affect the structures produced. A number of computational models for DNA-functionalised systems have successfully described their properties, and molecular simulation techniques have provided a unique insight into the factors underlying their assembly. Here, we discuss a variety of efforts using simulations to solve an important design problem in DNA-mediated assembly: how the properties of individual DNA-functionalised particles affect their interactions with each other, and ultimately how these interactions determine what structures can be produced. [ABSTRACT FROM AUTHOR]

Macromolecular crowding in cells is recognized to have a significant impact on biological function, yet quantitative models for its effects are relatively undeveloped. The influence of crowding on protein-protein interactions is of particular interest, since these mediate many processes in the cell, including the self-assembly of larger complexes, recognition, and signaling. We use a residue-level coarse-grained model to investigate the effects of macromolecular crowding on the assembly of protein-protein complexes. Interactions between the proteins are treated using a fully transferable energy function, and interactions of protein residues with the spherical crowders are repulsive. We show that the binding free energy for two protein complexes, ubiquitin/UIM1 and cytochrome c/cytochrome c peroxidase, decreases modestly as the concentration of crowding agents increases. To obtain a quantitative description of the stabilizing effect, we map the aspherical individual proteins and protein complexes onto spheres whose radii are calculated from the crowder-excluded protein volumes. With this correspondence, we find that the change in the binding free energy due to crowding can be quantitatively described by the scaled particle theory model without any fitting parameters. The effects of a mixture of different-size crowders-as would be found in a real cell-are predicted by the same model with an additivity ansatz. We also obtain the remarkable result that crowding increases the fraction of specific complexes at the expense of nonspecific transient encounter complexes in a crowded environment. This result, due to the greater excluded volume of the nonspecific complexes, demonstrates that macromolecular crowding can have subtle functional effects beyond the relative stability of bound and unbound complexes. [ABSTRACT FROM AUTHOR]

Many thermodynamic and dynamic properties of water display unusual behavior at low enough temperatures. In a recent study, Yan et al. [Phys. Rev. Lett. 95, 130604 (2005)] identified a spherically symmetric two-scale potential that displays many of the same anomalous properties as water. More specifically, for select parametrizations of the potential, one finds that the regions where isothermal compression anomalously (i) decreases the fluid’s structural order, (ii) increases its translational self-diffusivity, and (iii) increases its entropy form nested domes in the temperature-density plane. These property relationships are similar to those found for more realistic models of water. In this work, the authors provide evidence that suggests that the anomalous regions specified above can all be linked through knowledge of the excess entropy. Specifically, the authors show how entropy scaling relationships developed by Rosenfeld [Phys. Rev. A 15, 2545 (1977)] can be used to describe the region of diffusivity anomalies and to predict the state conditions for which anomalous viscosity and thermal conductivity behavior might be found. [ABSTRACT FROM AUTHOR]

TAR DNA-binding protein 43 (TDP-43) is involved in key processes in RNA metabolism and is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone, can undergo liquid-liquid phase separation (LLPS) in isolation, and is critical for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (QN-rich, IDR1/2) are also found to play a critical role in PS and aggregation. Compared with other phase-separating proteins, TDP-43 CTD has a notably distinct sequence composition including many aliphatic residues such as methionine and leucine. Aliphatic residues were previously suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution toward CTD phase separation has been relatively ignored. Using multiscale simulations coupled with in vitro saturation concentration (csat) measurements, we identified the importance of aromatic residues while also suggesting an essential role for aliphatic methionine residues in promoting single-chain compaction and LLPS. Surprisingly, NMR experiments showed that transient interactions involving phenylalanine and methionine residues in the disordered flanking regions can directly enhance site-specific, CR-mediated intermolecular association. Overall, our work highlights an underappreciated mode of biomolecular recognition, wherein both transient and site-specific hydrophobic interactions act synergistically to drive the oligomerization and phase separation of a disordered, low-complexity domain. [ABSTRACT FROM AUTHOR]

Experiments measuring contact formation between probes in disordered chains provide information on the fundamental time scales relevant to protein folding. However, their interpretation usually relies on one-dimensional (1D) diffusion models, as do many experiments probing a single distance. Here, we use all-atom molecular simulations to capture both the time scales of contact formation, as well as the scaling with peptide length for tryptophan triplet quenching experiments, revealing the sensitivity of the experimental quenching times to the configurational space explored by the chain. We find a remarkable consistency between the results of the full calculation and from Szabo-Schulten-Schulten theory applied to a 1D diffusion model, supporting the validity of such models. The significant reduction in diffusion coefficient at the small probe separations which most influence quenching rate, suggests that contact formation and Förster resonance energy transfer correlation experiments provide complementary information on diffusivity. [ABSTRACT FROM AUTHOR]

Amyloid aggregates are characteristic of many serious diseases such as Alzheimer's disease, Parkinson's, and type 2 diabetes and commonly involve intrinsically disordered proteins (IDPs), those that populate an ensemble of conformations rather than a single folded structure. Human islet amyloid polypeptide (hIAPP or amylin) is an amyloidogenic IDP implicated in pancreatic β-cell death during the pathogenesis of type 2 diabetes. The target of amylin's toxic activity is thought to be the cell's lipid membrane, which may also act as a catalyst for aggregation. Since amylin is intrinsically disordered, differing environments can have a large impact on its equilibrium conformational ensemble. We apply atomistic molecular dynamics simulations on multiple systems containing a full-length amylin monomer and a lipid bilayer to study the changes induced by the membrane. We observe stabilized helical conformations structurally similar to those determined by NMR experiments conducted in similar environments. We also find that bilayers of different compositions result in greatly different equilibrium ensembles of amylin. Finally, we discuss how a mixed bilayer containing zwitterionic and anionic lipid headgroups can allow for greater preference toward conformations which are adsorbed below the membrane surface through rearrangement of lipids for more favorable protein-lipid interactions. [ABSTRACT FROM AUTHOR]

Summary RNA-binding protein TDP-43 mediates essential RNA processing but forms cytoplasmic neuronal inclusions via its C-terminal domain (CTD) in amyotrophic lateral sclerosis (ALS). It remains unclear if aggregated TDP-43 is neurotoxic and if ∼50 ALS-associated missense mutations in TDP-43 CTD promote aggregation, or if loss of normal function plays a role in disease. Recent work points to the ability of related proteins to assemble into functional phase-separated ribonucleoprotein granules via their structurally disordered prion-like domains. Here, we provide atomic details on the structure and assembly of the low-complexity CTD of TDP-43 into liquid-liquid phase-separated in vitro granules and demonstrate that ALS-associated variants disrupt interactions within granules. Using nuclear magnetic resonance spectroscopy, simulation, and microscopy, we find that a subregion cooperatively but transiently folds into a helix that mediates TDP-43 phase separation. ALS-associated mutations disrupt phase separation by inhibiting interaction and helical stabilization. Therefore, ALS-associated mutations can disrupt TDP-43 interactions, affecting function beyond encouraging aggregation. [ABSTRACT FROM AUTHOR]

The misfolding and mutation of Cu/Zn superoxide dismutase (SOD1) is commonly associated with amyotrophic lateral sclerosis (ALS). SOD1 can accumulate within stress granules (SGs), a type of membraneless organelle, which is believed to form via liquid–liquid phase separation (LLPS). Using wild‐type, metal‐deficient, and different ALS disease mutants of SOD1 and computer simulations, we report here that the absence of Zn leads to structural disorder within two loop regions of SOD1, triggering SOD1 LLPS and amyloid formation. The addition of exogenous Zn to either metal‐free SOD1 or to the severe ALS mutation I113T leads to the stabilization of the loops and impairs SOD1 LLPS and aggregation. Moreover, partial Zn‐mediated inhibition of LLPS was observed for another severe ALS mutant, G85R, which shows perturbed Zn‐binding. By contrast, the ALS mutant G37R, which shows reduced Cu‐binding, does not undergo LLPS. In addition, SOD1 condensates induced by Zn‐depletion exhibit greater cellular toxicity than aggregates formed by prolonged incubation under aggregating conditions. Overall, our work establishes a role for Zn‐dependent modulation of SOD1 conformation and LLPS properties that may contribute to amyloid formation. Synopsis: The formation of membraneless organelles, such as stress granules (SGs), is believed to occur through the process of liquid–liquid phase separation (LLPS). The amyotrophic lateral sclerosis (ALS) disease‐associated protein SOD1 is known to accumulate within SGs, but if it itself undergoes LLPS, and if this affects amyloid formation, is not well‐studied. The apo form of SOD1 undergoes LLPS.SOD1 LLPS and subsequent aggregation are regulated by a conformational transition between a disordered and a relatively compact folded state.The metal cofactor Zn prevents SOD1 LLPS via a conformational transition.SOD1 condensates show greater cytotoxicity than aggregates. [ABSTRACT FROM AUTHOR]

We use all-atom molecular simulationwith explicit solvent to studythe properties of selected intrinsically disordered proteins and unfoldedstates of foldable proteins, which include chain dimensions and shape,secondary structure propensity, solvent accessible surface area, andcontact formation. We find that the qualitative scaling behavior ofthe chains matches expectations from theory under ambient conditions.In particular, unfolded globular proteins tend to be more collapsedunder the same conditions than charged disordered sequences of thesame length. However, inclusion of explicit solvent in addition naturallycaptures temperature-dependent solvation effects, which results inan initial collapse of the chains as temperature is increased, inqualitative agreement with experiment. There is a universal originto the collapse, revealed in the change of hydration of individualresidues as a function of temperature: namely, that the initial collapseis driven by unfavorable solvation free energy of individual residues,which in turn has a strong temperature dependence. We also observethat in unfolded globular proteins, increased temperature also initiallyfavors formation of native-like (rather than non-native-like) structure.Our results help to establish how sequence encodes the degree of intrinsicdisorder or order as well as its response to changes in environmentalconditions. [ABSTRACT FROM AUTHOR]

Thecholesterol recognition/interaction amino acid consensus (CRAC)motif is a primary structure pattern used to identify regions thatmay be responsible for preferential cholesterol binding in many proteins.The leukotoxin LtxA, which is produced by a pathogenic bacterium,contains two CRAC seqences, only one of which is responsible for cholesterolbinding, and the binding is required for cytotoxicity. The factors,in addition to the CRAC definition, that may be responsible for cholesterol-bindingfunctionality and atomistic interactions between the CRAC region andcholesterol are as yet unknown. This study uses molecular dynamicssimulations to identify structural characteristics and specific interactionsof the two LtxA CRAC peptides with both pure phospholipid and binarycholesterol–phospholipid bilayers. We have identified changesin the secondary structure of these peptides that occur upon cholesterolbinding, which are not seen when it is associated with a cholesterol-devoidmembrane, and which show salient coupling of structural disorder andfunction. Additionally, the central tyrosine residue of the CRAC motifwas found to play a significant role in cholesterol binding, thoughresidues outside of the CRAC motif also influence membrane interactionsand functionality of the CRAC region. [ABSTRACT FROM AUTHOR]

Replicaexchange molecular dynamics simulations are performed onthe protein complex pKID–KIX to understand the effects of macromolecularcrowding on coupled folding and binding events. A structure-basedprotein model at the residue level is adopted for the two proteinsto include intramolecular conformational flexibility, while crowdingmacromolecules are represented as spherical particles. The interactionsbetween crowders and protein residues can be either purely repulsiveor a combination of short-range repulsion and intermediate-range attraction.Consistent with previous studies on rigid-body protein binding inthe presence of spherical crowders, we find that the complex formationis stabilized by repulsive protein–crowder interactions anddestabilized by sufficiently strong attractive protein–crowderinteractions. Competition between stabilizing repulsive and destabilizingattractive interactions is quantitatively captured by a previous theoreticalmodel developed for describing the change in the binding free energyof rigid proteins in a crowded environment. We find that protein flexibilityhas little effect on the thermodynamics of the pKID–KIX binding(with respect to bulk) for repulsive and weakly attractive protein–crowderinteractions. For strong protein–crowder attractive interactions,the destabilizing effect due to crowding is attenuated by proteinflexibility. Interestingly, the mechanism of coupled folding and bindingobserved in bulk remains unchanged under highly crowded conditionsover a broad range of protein–crowder interaction strengths.Also, strong protein–crowder attractive interactions can significantlystabilize intermediate states involving partial contact between pKIDand KIX domains. [ABSTRACT FROM AUTHOR]

We perform molecular dynamics simulations to study the transport of geometrically modified water models through channels of carbon nanotube (CNT) membranes. We use two modifications to an existing water model (extended simple point charge SPC/E) as representative surrogates of molecular fluids: (1) bent model (model B) in which the HOH angle is varied while keeping the dipole moment constant by adjusting the OH bond length and (2) modified bent model (model MB) in which the HOH angle changes without any change in OH bond length thereby changing the dipole moment. Interestingly, we find that the fluid transport is a nonmonotonic function of the bond angle for both fluid models. This observed trend is not anticipated based on the fluid density as a function of the bond angle inside and outside of the nanotube channel. However, the average residence time of transmitted molecules through the channel provides an approximately inverse linear correlation with the observed flux, independent of the fluid model. Based on these correlations, we have developed an empirical design parameter connecting fluid transport through CNTs as a function of average occupancy (number of fluid molecules inside the nanotube) and the average residence time. Our results suggest that transport through carbon nanotubes can be sensitive to small changes in the structure of fluid molecules that can potentially be utilised for mixture separation. [ABSTRACT FROM PUBLISHER]

Formation of membrane‐less organelles by self‐assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more β‐sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications. [ABSTRACT FROM AUTHOR]

Formation of membrane‐less organelles by self‐assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more β‐sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications. [ABSTRACT FROM AUTHOR]

Formation of membrane‐less organelles by self‐assembly of disordered proteins can be triggered by external stimuli such as pH, salt, or temperature. These organelles, called biomolecular condensates, have traditionally been classified as liquids, gels, or solids with limited subclasses. Here, the authors show that a thermal trigger can lead to formation of at least two distinct liquid condensed phases of the fused in sarcoma low complexity (FUS LC) domain. Forming FUS LC condensates directly at low temperature leads to formation of metastable, kinetically trapped condensates that show arrested coalescence, escape from which to untrapped condensates can be achieved via thermal annealing. Using experimental and computational approaches, the authors find that molecular structure of interfacial FUS LC in kinetically trapped condensates is distinct (more β‐sheet like) compared to untrapped FUS LC condensates. Moreover, molecular motion within kinetically trapped condensates is substantially slower compared to that in untrapped condensates thereby demonstrating two unique liquid FUS condensates. Controlling condensate thermodynamic state, stability, and structure with a simple thermal switch may contribute to pathological protein aggregate stability and provides a facile method to trigger condensate mixing for biotechnology applications. [ABSTRACT FROM AUTHOR]

Human islet amyloid polypeptide (hIAPP),a 37-residue protein cosecretedwith insulin by β-cells in the pancreas, is known to form amyloidfibrils in type II diabetes patients. This fibril formation is alsoassociated with β-cell death. However, rat IAPP (rIAPP) doesnot aggregate into fibrils, nor is it associated with β-celltoxicity. Determining solutionproperties of hIAPP experimentally is difficult because it aggregatesquickly. Our study uses molecular dynamics simulation to explore andcompare in-solution characteristics of hIAPP and rIAPP, as well astwo single-point hIAPP mutants, hIAPP I26P and hIAPP S20G, which exhibitobserved differences from hIAPP in aggregation propensities. We findthat all four polypeptide monomers sample structured states in solution.More importantly, differences in the helicity over residues 7–16may play an important role in early aggregation, although this regionis outside of commonly assumed amyloidogenic region 20–29.The long-range contacts, though unexpected of IDPs, cause variationin sampled conformations among four polypeptides within same aminoacid sequence. Our results also yield evidence that previously determinedstructures bound to micelles are also transiently sampled in the solutionstate. In particular, similarities found in region 8–17 togetherwith the helical differences that we observe in region 7–16lead us to suggest that the region 7–16 is potentially responsiblefor amyloidogenic behavior of amylin peptides. Our results also providesupport for the proposed mechanism of fibril formation based on experimentallyobserved transient helices in amyloidogenic peptides. [ABSTRACT FROM AUTHOR]

Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase-separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates. [ABSTRACT FROM AUTHOR]

The single-stranded DNA to single-walled carbon nanotube(SWCNT)hybrid continues to attract significant interest as an exemplary biologicalmolecule–nanomaterial conjugate. In addition to their manybiomedical uses, such as in vivo sensing and delivery of molecularcargo, DNA-SWCNT hybrids enable the sorting of SWCNTs according totheir chirality. Current experimental methods have fallen short ofidentifying the actual structural ensemble of DNA adsorbed onto SWCNTsthat enables and controls several of these phenomena. Molecular dynamics(MD) simulation has been a useful tool for studying the structureof these hybrid molecules. In recent studies, using replica exchangeMD (REMD) simulation we have shown that novel secondary structuresemerge and that these structures are DNA-sequence and SWCNT-type dependent.Here, we use REMD to investigate in detail the structural characteristicsof two DNA-SWCNT recognition pairs: (TAT)4-(6,5)-SWCNT,i.e., DNA sequence TATTATTATTAT bound to the (6,5)chirality SWCNT, and (CCG)2CC-(8,7)-SWCNT as well as off-recognitionpairs (TAT)4-(8,7)-SWCNT and (CCG)2CC-(6,5)-SWCNT.From a structural clustering analysis, dominant equilibrium structuresare identified and show a right-handed self-stitched motif for (TAT)4-(6,5) in contrast to a left-handed β-barrel for (CCG)2CC-(8,7). Additionally, characteristics such as DNA end-to-enddistance, solvent accessible SWCNT surface area, DNA hydrogen bondingbetween bases, and DNA dihedral distributions have been probed indetail as a function of the number of DNA strands adsorbed onto thenanotube. We find that the DNA structures adsorbed onto a nanotubeare also stabilized by significant numbers of non-Watson–Crickhydrogen bonds (intrastrand and interstrand) in addition to π–πstacking between DNA bases and nanotube surface and Watson–Crickpairs. Finally, we provide a summary of DNA structures observed forvarious DNA-SWCNT hybrids as a preliminary set of motifs that maybe involved in the functional role of these hybrids. [ABSTRACT FROM AUTHOR]

Single- and double-stranded DNA are increasingly beingpaired withsurfaces and nanoparticles for numerous applications, such as sensing,imaging, and drug delivery. Unlike the majority of DNA structuresin bulk that are stabilized by canonical Watson–Crick pairingbetween Ade-Thy and Gua-Cyt, those adsorbed on surfaces are oftenstabilized by noncanonical base pairing, quartet formation, and base–surfacestacking. Not much is known about these kinds of interactions. Tobuild an understanding of the role of non-Watson–Crick pairingon DNA behavior near surfaces, one requires basic information on DNAbase pair stacking and hydrogen-bonding interactions. All-atom molecularsimulations of DNA bases in two casesin bulk water and stronglyadsorbed on a graphite surfaceare conducted to study the relativestrengths of stacking and hydrogen bond interactions for each of the10 possible combinations of base pairs. The key information obtainedfrom these simulations is the free energy as a function of distancebetween two bases in a pair. We find that stacking interactions exertthe dominant influence on the stability of DNA base pairs in bulkwater as expected. The strength of stability for these stacking interactionsis found to decrease in the order Gua-Gua > Ade-Gua > Ade-Ade> Gua-Thy> Gua-Cyt > Ade-Thy > Ade-Cyt > Thy-Thy > Cyt-Thy >Cyt-Cyt. On theother hand, mutual interactions of surface-adsorbed base pairs arestabilized mostly by hydrogen-bonding interactions in the order Gua-Cyt> Ade-Gua > Ade-Thy > Ade-Ade > Cyt-Thy > Gua-Gua >Cyt-Cyt > Ade-Cyt> Thy-Thy > Gua-Thy. Interestingly, several non-Watson–Crickbase pairings, which are commonly ignored, have similar stabilizationfree energies due to interbase hydrogen bonding as Watson–Crickpairs. This clearly highlights the importance of non-Watson–Crickbase pairing in the development of secondary structures of oligonucleotidesnear surfaces. [ABSTRACT FROM AUTHOR]

The DNA-single-walled carbon nanotube (SWCNT) hybrid molecule has attracted signfficant attention recently for its ability to disperse and sort SWCNTs according to their chirality. Key for utilizing their unique properties is an understanding of the structure of DNA adsorbed on the SWCNT surface, which we study here using molecular simulations. Using replica exchange molecular dynamics (REMD), we explore equilibrium structures formed by single strands of 12-mer oligonucleotides, of varying sequence, adsorbed on a (6,5)-SWCNT. We find a consistent motif in which the DNA strand forms a right-handed helical wrap around the SWCNT, stabilized by "stitches" (hydrogen bonding between distant bases) to itself. Variability among equilibrium populations of DNA self-stitched structures was observed and shown to be directly influenced by DNA sequence and composition. Competition between conformational entropy and hydrogen bonding between bases is predicted to be responsible for the formation of random versus stitched configurations. [ABSTRACT FROM AUTHOR]

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity. SYNOPSIS: Specificity of contacts formed in phase‐separated RNA granules is complex. We show phosphorylation alters hnRNPA2 interactions and expression of activated Fyn kinase prevents neurodegeneration, suggesting a path to therapies for aggregation diseases. hnRNPA2 granule components hnRNPF and TOG specifically partition into hnRNPA2 low complexity domain droplets.Tyrosine phosphorylation prevents this partitioning as well as aggregation of hnRNPA2 disease‐associated mutants.A C. elegans model of ALS/FTD associated with hnRNPA2‐D290V shows glutamatergic neurodegeneration.Deletion of tdp‐1 (TDP‐43 ortholog) or expression of activated Fyn (tyrosine kinase that phosphorylates hnRNPA2) reduces neurodegeneration. [ABSTRACT FROM AUTHOR]

Phase 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 bioinspired 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. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. 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 despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation. [ABSTRACT FROM AUTHOR]

Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS. [ABSTRACT FROM AUTHOR]

A correction to the article "Excess-entropy-based anomalies for a waterlike fluid" that appeared in a previous issue of this periodical is presented.