Macromolecular Crowding Research Articles

Roberge-Weiss periodicity and singularity in a hadron resonance gas model with excluded volume effects

Quantum chromodynamics (QCD) with pure imaginary baryon number chemical potential μ=iθT, where T is temperature and θ is a real number, has the Roberge-Weiss periodicity. We study the θ-dependence of the baryon number density and the pressure in the hadron resonance gas model with excluded volume effects of baryons. It is shown that the baryon number density and the pressure are smooth periodic functions of θ at low or high temperature. However, they have singular behavior at θ=(2k+1)π where k is an integer, when T∼211 MeV. This temperature is consistent with the Roberge-Weiss transition temperature TRW obtained by lattice QCD simulations. This singularity can be explained by the dual excluded volume effects in which the roles of pointlike and nonpointlike particles are exchanged each other in the ordinary excluded volume effects. It is also indicated that the excluded volume effect is visible just below TRW and is directly detectable by the lattice QCD simulation at finite θ. We compare the results with the one obtained by the Polyakov-loop extended Nambu–Jona-Lasinio model. Published by the American Physical Society 2025

The home advantage and COVID-19: the crowd support effect on the english football premier league and the championship

Abstract It is well known that there is an home advantage in football (American English: soccer) where the home team wins in about 45% of the games compared to the 27% of the away team. This has mainly been attributed to the support of the home audience and is commonly denoted the crowd support effect. The COVID-19 pandemic forced many football leagues to play the games without spectators thus making it possible to analyse the effect of crowd support in football. We analyse more than 18,000 games in the two top English football leagues during the period 2001–2020 for the Premier league and the Championship with an ordinal logistic model with explanatory variables (e.g., previous team performance, a time trend, league dummy) including a pandemic dummy. We discovered that the absence of spectators has no impact on the outcome probability in the Premier League. However, it significantly reduces the probability of the home team wins in the Championship.

Osmotic stress influences microtubule drug response via WNK1 kinase signaling.

Ion homeostasis is critical for numerous cellular processes, and disturbances in ionic balance underlie diverse pathological conditions, including cancer progression. Targeting ion homeostasis is even considered as a strategy to treat cancer. However, very little is known about how ion homeostasis may influence anticancer drug response. In a genome-wide CRISPR-Cas9 resistance drug screen, we identified and validated the master osmostress regulator WNK1 kinase as a modulator of the response to the mitotic inhibitor rigosertib. Osmotic stress and WNK1 inactivation lead to an altered response not only to rigosertib treatment but also to other microtubule-related drugs, minimizing the prototypical mitotic arrest produced by these compounds. This effect is due to an alteration in microtubule stability and polymerization dynamics, likely maintained by fluctuations in intracellular molecular crowding upon WNK1 inactivation. This promotes resistance to microtubule depolymerizing compounds, and increased sensitivity to microtubule stabilizing drugs. In summary, our data proposes WNK1 osmoregulation activity as an important modulator for microtubule-associated chemotherapy response.

Dynamical arrest for globular proteins with patchy attractions.

Attempts to use colloid science concepts to better understand the dynamic properties of concentrated or crowded protein solutions are challenging due to the fact that globular proteins generally have heterogeneous surfaces that result in anisotropic or patchy contributions to their interaction potential. This is particularly difficult when targeting non-equilibrium transitions such as glass and gel formation in concentrated protein solutions. Here we report a systematic study of the reduced zero shear viscosity ηr of the globular protein γB-crystallin, an eye lens protein that plays a vital role in vision-related phenomena such as cataract formation or presbyopia, and compare the results to the existing structural and dynamic data. Combining two different tracer particle-based microrheology methods allows us to precisely locate the line of kinetic arrest within the phase diagram and characterize the functional form of the concentration and temperature dependence of ηr. We show that while our results qualitatively confirm the existing view that this protein can be reasonably well described using a coarse-grained picture of a patchy colloid with short range attractions, there are a number of novel findings that cannot easily be understood with the existing simple colloid models. We demonstrate in particular the complete failure of an extended law of corresponding states for a description of the temperature dependence of the arrest line, and discuss the role that transient clusters play in this context.

Protocols for translocation processes of flexible polymers through a pore using LAMMPS.

Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) is an open-source, powerful simulator with a customizable platform for extensive Langevin dynamics simulations. Here, we present a protocol for using LAMMPS to develop coarse-grained models of polymeric systems with macromolecular crowding, an integral part of any soft matter or biophysical system. We describe steps for installing software, using LAMMPS basic commands and code, and translocating polymers. This protocol has potential applications in developing mathematical models for DNA sequencing, controlled drug delivery, and cellular transport processes. For complete details on the use and execution of this protocol, please refer to Garg etal.1.

A Combined Approach to Extract Rotational Dynamics of Globular Proteins Undergoing Liquid-Liquid Phase Separation.

The formation of protein condensates (droplets) via liquid-liquid phase separation (LLPS) is a commonly observed phenomenon in vitro. Changing the environmental properties with cosolutes, molecular crowders, protein partners, temperature, pressure, etc. has been shown to favor or disfavor the formation of protein droplets by fine-tuning the water-water, water-protein, and protein-protein interactions. Therefore, these environmental properties and their spatiotemporal fine-tuning are likely to be important also in a cellular context at the existing protein expression levels. One of the key physicochemical properties of biomolecules impacted by molecular crowding is diffusion, which determines the viscoelastic behavior of the condensates. Here, we investigate the change in the rotational diffusion of γD-crystallin, undergoing LLPS in vitro in aqueous solutions in the absence and presence of cosolutes. We studied its rotational dynamics using molecular dynamics simulations (MD), electron paramagnetic resonance (EPR) spectroscopy, and fluorescence spectroscopy. MD simulations performed under dilute and crowded conditions show that the rotational diffusion of crystallin in water is retarded by 1 to 2 orders of magnitude in the condensed phase. To obtain the rotational dynamics in the dilute phase, we used fluorescence anisotropy and to extract the retardation factor in the condensed phase, we used spin-labeled γD-crystallin proteins as EPR viscosity nanoprobes. Aided by a viscosity nanoruler calibrated with solutions at increasing sucrose concentrations, we validated the rotational diffusion retardation predicted by MD simulations. This study underlines the predictive power of MD simulations and showcases the use of a sensitive EPR nanoprobe to extract the viscosity of biomolecular condensates.

Influence of Magnesium Ions and Crowding Agents on Structure and Stability of RNA Aptamers.

Aptamers bind to their targets with exceptional affinity and specificity. However, their intracellular application is hampered by the lack of knowledge about the effect of the cellular milieu on the RNA structure/stability. In this study, cellular crowding was mimicked using polyethylene glycol (PEG), and the crucial role of Mg2+ ions in stabilizing the structure of an RNA aptamer was investigated. Increasing the concentration of Mg2+ or PEG increased the thermal stability of the aptamer. The crowding effect lowered the requirement of the Mg2+ ion to form the binding-competent conformer of the aptamer. This suggests that crowding and other factors may compensate for a lower concentration of Mg2+ for proper folding of aptamers inside cells. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing permitted residue-level analysis of the aptamer. Mg2+ and/or PEG were shown to be involved in increasing the rigidity or flexibility of different regions of the aptamer. Fluorescence correlation spectroscopy showed a significantly low hydrodynamic radius (RH) in the presence of molecular crowders and Mg2+ ions. We believe that the decreased water activity due to crowding may be responsible for reduced RH. Our results show that in a crowded environment, the RNA aptamer was exposed to conformers that were not available to it in simple buffer solutions or solely in the presence of lower concentrations of Mg2+.

Tuning the Crowding Effect of Water and Imidazole in Covalent Organic Frameworks for Proton Conduction.

The proton conduction of imidazole under confined conditions has attracted widespread attention from researchers. Under anhydrous conditions, the proton transfer behavior is primarily governed by the molecular dynamics of imidazole. However, within a water-mediated system, the crowding effect of water and imidazole in a confined space may outweigh the intrinsic properties of imidazole itself. In this study, we have meticulously adjusted the structural fragments within the covalent organic frameworks (COFs), fine-tuning the saturation level of imidazole loading and adjusting the crowding degree of imidazole and water molecules. As a result, the two COF composites exhibit distinctly different proton conduction mechanisms from 32 to 100% relative humidity (RH), of which one possesses proton conduction progressively shifting from the Grotthuss mechanism to the vehicle mechanism, while the other has proton conduction undergoing a transition from the vehicle mechanism at 32% RH through the Grotthuss mechanism at 75% RH and finally back to the vehicle mechanism at 100% RH. These results highlight the critical role of the crowding effect of water and imidazole within confined spaces in proton conduction.

Visual attention matters during word recognition: A Bayesian modeling approach.

It is striking that visual attention, the process by which attentional resources are allocated in the visual field so as to locally enhance visual perception, is a pervasive component of models of eye movements in reading, but is seldom considered in models of isolated word recognition. We describe BRAID, a new Bayesian word-Recognition model with Attention, Interference and Dynamics. As most of its predecessors, BRAID incorporates three sensory, perceptual, and orthographic knowledge layers together with a lexical membership submodel. Its originality resides in also including three mechanisms that modulate letter identification within strings: an acuity gradient, lateral interference, and visual attention. We calibrated the model such that its temporal scale was consistent with behavioral data, and then explored the model's capacity to generalize to other, independent effects. We evaluated the model's capacity to account for the word length effect in lexical decision, for the optimal viewing position effect, and for the interaction of crowding and frequency effects in word recognition. We further examined how these effects were modulated by variations in the visual attention distribution. We show that visual attention modulates all three effects and that a narrow distribution of visual attention results in performance patterns that mimic those reported in impaired readers. Overall, the BRAID model could be conceived as a core building block, towards the development of integrated models of reading aloud and eye movement control, or of visual recognition of impaired readers, or any context in which visual attention does matter.

The Balbiani body is formed by microtubule-controlled molecular condensation of Buc in early oogenesis.

Vertebrate oocyte polarity has been observed for two centuries and is essential for embryonic axis formation and germline specification, yet its underlying mechanisms remain unknown. In oocyte polarization, critical RNA-protein (RNP) granules delivered to the oocyte's vegetal pole are stored by the Balbiani body (Bb), a membraneless organelle conserved across species from insects to humans. However, the mechanisms of Bb formation are still unclear. Here, we elucidate mechanisms of Bb formation in zebrafish through developmental biomolecular condensation. Using super-resolution microscopy, live imaging, biochemical, and genetic analyses invivo, we demonstrate that Bb formation is driven by molecular condensation through phase separation of the essential intrinsically disordered protein Bucky ball (Buc). Live imaging, molecular analyses, and fluorescence recovery after photobleaching (FRAP) experiments invivo reveal Buc-dependent changes in the Bb condensate's dynamics and apparent material properties, transitioning from liquid-like condensates to a solid-like stable compartment. Furthermore, we identify a multistep regulation by microtubules that controls Bb condensation: first through dynein-mediated trafficking of early condensing Buc granules, then by scaffolding condensed granules, likely through molecular crowding, and finally by caging the mature Bb to prevent overgrowth and maintain shape. These regulatory steps ensure the formation of a single intact Bb, which is considered essential for oocyte polarization and embryonic development. Our work offers insight into the long-standing question of the origins of embryonic polarity in non-mammalian vertebrates, supports a paradigm of cellular control over molecular condensation by microtubules, and highlights biomolecular condensation as a key process in female reproduction.

The Effects of Crowds on Professional European Basketball Teams

Do crowds affect professional basketball teams’ performance? Using results from matches played in the EuroLeague, EuroCup, and ACB (Spain) before, during, and after the COVID period (2014–2024) allows us to answer this question. Our estimations conclude that the probability of a team winning at home is 5.6% lower in the absence of a home crowd in ACB and 5.76% in the case of EuroLeague. Moreover, the difference between home points and away points is lower and the percentage home fouls versus away fouls is higher, but both results are only significant for ACB. Finally, the absence of spectators in the stands does not affect shooting percentages.

Impact of physiological ionic strength and crowding on kinesin-1 motility.

The motility of biological molecular motors has typically been analyzed by in vitro reconstitution systems using motors isolated and purified from organs or expressed in cultured cells. The behavior of biomolecular motors within cells has frequently been reported to be inconsistent with that observed in reconstituted systems in vitro. Although this discrepancy has been attributed to differences in ionic strength and intracellular crowding, understanding how such parameters affect the motility of motors remains challenging. In this report, we investigated the impact of intracellular crowding in vitro on the mechanical properties of kinesin under a high ionic strength that is comparable to the cytoplasm. Initially, we characterized viscosity in a cell by using a kinesin motor lacking the cargo-binding domain. We then used polyethylene glycol to create a viscous environment in vitro comparable to the intracellular environment. Our results showed that kinesin frequently dissociated from microtubules under high ionic strength conditions. However, under conditions of both high ionic strength and crowding with polymers, the processive movement of kinesin persisted and increased in frequency. This setting reproduces the significant variations in the mechanical properties of motors measured in the intracellular environment and suggests a mechanism whereby kinesin maintains motility under the high ionic strengths found in cells.Key words: Kinesin motility, molecular crowding, ionic strength, intracellular transport, processivity of molecular motors.

Time-resolved fluorescence of ANS dye as a sensor of proteins LLPS.

The explosive growth in the number of works addressing the phase separation of intrinsically disordered proteins has driven both the development of new approaches and the optimization of existing methods for biomolecular condensate visualization. In this work, we studied the potential use of the fluorescent dye ANS as a sensor for liquid-liquid phase separation (LLPS), focusing on visualizing condensates formed by the stress-granules scaffold protein G3BP1. Using fluorescence lifetime imaging microscopy (FLIM), we demonstrated that ANS can accumulate in RNA-induced G3BP1 condensates in aqueous solutions, but not in G3BP1 condensates formed under macromolecular crowding conditions in highly concentrated PEG solutions. We showed that the experimentally determined limiting fluorescence anisotropy (r0'), which characterizes the amplitude of high-frequency intramolecular mobility of ANS in aqueous solutions containing RNA-induced G3BP1 condensates, is half the value observed for ANS in aqueous G3BP1 solutions. Our results demonstrate the feasibility of using time-resolved fluorescence spectroscopy and microscopy of ANS for detecting LLPS of intrinsically disordered proteins in aqueous solutions.

Binding and stabilizing effects of ruthenium(II) polypyridyl complexes [Ru(bpy)2(6-L-dppz)]2+ (L = F and CH3) toward duplex RNA poly(A)·poly(U) under molecular crowding conditions

Binding and stabilizing effects of ruthenium(II) polypyridyl complexes [Ru(bpy)2(6-L-dppz)]2+ (L = F and CH3) toward duplex RNA poly(A)·poly(U) under molecular crowding conditions

Random walk models in the life sciences: including births, deaths and local interactions.

Random walks and related spatial stochastic models have been used in a range of application areas, including animal and plant ecology, infectious disease epidemiology, developmental biology, wound healing and oncology. Classical random walk models assume that all individuals in a population behave independently, ignoring local physical and biological interactions. This assumption simplifies the mathematical description of the population considerably, enabling continuum-limit descriptions to be derived and used in model analysis and fitting. However, interactions between individuals can have a crucial impact on population-level behaviour. In recent decades, research has increasingly been directed towards models that include interactions, including physical crowding effects and local biological processes such as adhesion, competition, dispersal, predation and adaptive directional bias. In this article, we review the progress that has been made with models of interacting individuals. We aim to provide an overview that is accessible to researchers in application areas, as well as to specialist modellers. We focus particularly on derivation of asymptotically exact or approximate continuum-limit descriptions and simplified deterministic models of mean-field behaviour and resulting spatial patterns. We provide worked examples and illustrative results of selected models. We conclude with a discussion of current areas of focus and future challenges.

Ultrahigh concentration exfoliation and aqueous dispersion of few-layer graphene by excluded volume effect

Ultrahigh concentration exfoliation and aqueous dispersion of few-layer graphene by excluded volume effect

How to Compute Density Fluctuations at the Nanoscale.

The standard definition of particle number fluctuations based on point-like particles neglects the excluded volume effect. This leads to a large and systematic finite-size scaling and an unphysical surface term in the isothermal compressibility. We correct these errors by introducing a modified pair distribution function that takes account of the finite size of the particles. For the hard sphere fluid in one-dimension, we show that the compressibility is strictly size-independent, and we reproduce this result from the number fluctuations calculated with the new theory. In general, the present method eliminates the leading finite-size effect, which makes it possible to compute density fluctuations accurately in very small sampling volumes, comparable to a single particle size. These findings open the way for obtaining the local compressibility from fluctuation theory at the nanometer scale.

Manipulating Toughness and Microstructure in Polyelectrolyte Complex Hydrogels with Competitive Surfactant Micelles.

Polyelectrolyte complex (PEC) hydrogels provide a promising strategy to develop a class of physically cross-linked networks characterized by exceptional toughness and self-healing properties. However, the precise control of the microstructure and the enhancement of mechanical properties still pose challenges in the field of PEC hydrogels. Herein, we propose a strategy to manipulate the structure of PEC with competitively charged surfactant micelles, leveraging the spatially confined surface charge and excluded volume effects to overcome coacervation issues associated with the PEC, thus achieving a simple one-step preparation of macroscopically uniform and tough PEC hydrogels. Specifically, polyelectrolyte complex/surfactant micelle (PEC-SM) hybrid hydrogels were prepared by one-step copolymerization of chitosan (CS)/acrylic acid/cetyltrimethylammonium bromide (CTAB) micelles. The content of CTAB micelles was found to continuously modulate both the structure and the mechanical properties of the resulting PEC-SM hydrogel network. On one hand, reversible deformation-recovery behavior exhibited by CTAB cavity micelles through hydrophobic interactions efficiently dissipates energy; on the other hand, competition between CS chains and CTAB micelles for electrostatic binding sites with poly(acrylic acid), along with excluded volume effects of CTAB micelles, imparts a hierarchical structure upon the PEC-SM hydrogel. Rheology provided detailed insights into the viscoelastic behaviors of PEC-SM hydrogels at varying CTAB concentrations. The intermolecular interaction and heterogeneous network structure of physically cross-linked PEC-SM hydrogels with CTAB micelles were elucidated by solid-state nuclear magnetic resonance (NMR) spectroscopy. On the basis of rheology and NMR results, complemented by other characterization analyses, the physical illustration of the PEC-SM hybrid hydrogel network structure regulated by competitive surfactant micelles is presented. This work offers valuable in-depth insight into polyelectrolyte complexation and provides a foundation for the development of robust PEC hydrogel materials.

Binding site maturation modulated by molecular density underlies Ndc80 binding to kinetochore receptor CENP-T

Macromolecular assembly depends on tightly regulated pairwise binding interactions that are selectively favored at assembly sites while being disfavored in the soluble phase. This selective control can arise due to molecular density-enhanced binding, as recently found for the kinetochore scaffold protein CENP-T. When clustered, CENP-T recruits markedly more Ndc80 complexes than its monomeric counterpart, but the underlying molecular basis remains elusive. Here, we use quantitative in vitro assays to reveal two distinct mechanisms driving this behavior. First, Ndc80 binding to CENP-T is a two-step process: initially, Ndc80 molecules rapidly associate and dissociate from disordered N-terminal binding sites on CENP-T. Over time, these sites undergo maturation, resulting in stronger Ndc80 retention. Second, we find that this maturation transition is regulated by a kinetic barrier that is sensitive to the molecular environment. In the soluble phase, binding site maturation is slow, but within CENP-T clusters, this process is markedly accelerated. Notably, the two Ndc80 binding sites in human CENP-T exhibit distinct maturation rates and environmental sensitivities, which correlate with their different amino acid content and predicted binding conformations. This clustering-induced maturation is evident in dividing human cells, suggesting a distinct regulatory entry point for controlling kinetochore assembly. We propose that the tunable acceleration of binding site maturation by molecular crowding may represent a general mechanism for promoting the formation of macromolecular structures.

Pitfalls of Using ANS Dye Under Molecular Crowding Conditions.

The 1-anilino-8-naphthalenesulfonate (ANS) fluorescent dye is widely used in protein folding studies due to the significant increase in its fluorescence quantum yield upon binding to protein hydrophobic regions that become accessible during protein unfolding. However, when modeling cellular macromolecular crowding conditions in protein folding experiments in vitro using crowding agents with guanidine hydrochloride (GdnHCl) as the denaturant, the observed changes in ANS spectral characteristics require careful consideration. This study demonstrates that crowding agents can form clusters that interact differently with ANS. Furthermore, GdnHCl can disrupt these clusters and directly affect the ANS spectral characteristics. A model for the interaction between GdnHCl, crowders, and ANS is proposed. Using bovine serum albumin (BSA) as a model protein, the limitations of using ANS for studying conformational transitions induced by GdnHCl in the presence of crowding agents are demonstrated.