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bioRxiv2024

Growth of Staphylococcus aureus in the presence of oleic acid shifts the glycolipid fatty acid profile and increases resistance to antimicrobial peptides

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When S. aureus is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids. However, when unsaturated fatty acids are present in the growth medium, they become a major part of the total fatty acid composition. This study explores the biophysical effects of incorporating straight-chain unsaturated fatty acids into S. aureus membrane lipids. Membrane preparations from cultures supplemented with oleic acid showed more complex differential scanning calorimetry scans than those grown in tryptic soy broth alone. When grown in the presence of oleic acid, the cultures exhibited a transition significantly above the growth temperature, attributed to the presence of glycolipids with long-chain fatty acids causing acyl chain packing frustration within the bilayer. Functional aspects of the membrane were assessed by studying the kinetics of dye release from unilamellar vesicles induced by the antimicrobial peptide mastoparan X. Dye release was slower from liposomes prepared from cells grown in oleic acid-supplemented cultures, suggesting that changes in membrane lipid composition and biophysics protect the cell membrane against peptide-induced lysis. These findings underscore the intricate relationship between the growth environment, membrane lipid composition, and the physical properties of the bacterial membrane, which should be considered when developing new strategies against S. aureus infections.

biophysics↗

bioRxiv2024

Mechanical Analysis of Cell Migration Using Hybrid Structural Optimization

Cell migration requires the dynamic formation and dissolution of mechanical structures inside the cytoplasm. Stress fibers are made of F-actin during cell migration driven by the strategic localization of focal adhesion complexes at the cell-substrate interface. The nucleus is also strategically positioned in the cell during the migration and the stress fibers wrap around the nucleus possibly to carry the nucleus with the cell. Cell migration is energetically demanding and should require strategic utilization of resources such as the F-actin stress fiber formation at specific locations so that they generate enough force by actomyosin contraction at the cell-matrix adhesion sites for a directed movement. In this work we propose a structural optimization based biophysical model to predict the strategic localization and sizes of F-actin fibers that supports the nucleus and the cytoplasm during migration. With the use of a nonlinear controller via a Newton-Euler-based model of the generated design, we further quantified the force in the stress fibers during migration, with results close to those obtained through experimental methods such as traction force microscopy. The predicted force decreases for a cell that migrates slowly due to a pharmacological perturbation. Such quantification of forces only require the information of the trajectory of the cell that can be readily obtained from time lapse microscopy. With novel microscopy techniques emerging, such biophysical model framework can be combined with traction force microscopy data to achieve unprecedented mechanical information inside and outside cells during migration, which is otherwise not possible by experiments only. SIGNIFICANCECell migration plays a critical role in biological functions. It requires the strategic formation of F-actin stress fibers at specific locations, to generate forces by actomyosin contraction for cells to migrate in a directed manner. The present study predicts the localization and force generated by stress fibers based on the trajectory of the cell, which can be obtained via time lapse microscopy. The technique can complement other techniques such as traction force microscopy to provide mechanical information inside and outside cells during cell migration.

biophysics↗

bioRxiv2024

Lipid oxidation alters membrane mechanics favoring permeabilization

Ferroptosis is a form of regulated necrosis characterized by the iron-dependent accumulation of lipid peroxides in cell membranes. However, how lipid oxidation affects biophysical properties of cellular membranes and how these changes contribute to the opening of membrane pores are major questions in the field. Therefore, understanding the interplay between oxidized phospholipids and alterations in membrane biophysical properties is essential to unravel the underlying mechanisms of ferroptosis. Here, we characterized membrane alterations upon lipid oxidation in in vitro model membrane systems using lipid vesicles and supported lipid bilayers (SLBs) in which lipid oxidation was induced by Fenton reactions. We find that vesicle permeabilization kinetically correlates with the appearance of malondialdehyde (MDA), a product of lipid oxidation. Lipid oxidation also alters the lateral organization of SLBs exhibiting liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence, reducing the lipid mismatch between domains and decreasing the circularity of Lo domains, indicating a decrease in line tension at the phase boundaries. Further characterization of SLBs by force spectroscopy reveals a significant reduction in the average membrane breakthrough force upon oxidation, indicating changes in lipid bilayer organization that make it more susceptible to permeabilization. Our findings suggest that lipid oxidation induces strong changes in membrane lipid interactions and in the mechanical properties of membranes leading to reduced tension in the permeabilized state of the bilayer, which promotes membrane pore formation.

biophysics↗

bioRxiv2024

Developing peptide-based fusion inhibitors as an antiviral strategy utilizing Coronin 1 as a template

Enveloped viruses can enter the host cells by endocytosis and subsequently fuse with the endosomal membranes, or fuse with the plasma membrane at the cell surface. The crucial stage of viral infection, regardless of the route taken to enter the host cell, is membrane fusion. The present work aims to develop a peptide-based fusion inhibitor that prevents membrane fusion by modifying the properties of the participating membranes, without targeting a protein. This would allow us to develop a fusion inhibitor that might work against a larger spectrum of enveloped viruses as it does not target any specific viral fusion protein. With this goal, we have designed a novel peptide by modifying a native sequence derived from coronin 1, a phagosomal protein, that helps to avoid lysosomal degradation of mycobacterium-loaded phagosomes. The designed peptide, mTG-23, inhibits [~] 30-40% fusion between small unilamellar vesicles containing varying amounts of cholesterol by modulating the biophysical properties of the participating bilayers. As a proof of principle, we have further demonstrated that the mTG-23 inhibits Influenza A virus infection in A549 and MDCK cells (with[~] EC50 of 20.45 {micro}M and 21.45 {micro}M, respectively), where viral envelope and endosomal membrane fusion is a crucial step. Through a gamut of biophysical and biochemical methods, we surmise that mTG-23 inhibits viral infection by inhibiting viral envelope and endosomal membrane fusion. We envisage that the proposed antiviral strategy can be extended to other viruses that employ a similar modus operandi, providing a novel pan-antiviral approach.

biophysics↗

bioRxiv2024

Reversible disulfide bond crosslinks as tunable levers of phase separation in designer biomolecular condensates.

Biomolecular condensates (BCs) are membraneless hubs enriched in proteins and nucleic acids that have become important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is important in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the last decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to determine the involvement of disulfide crosslinks and their redox sensitivity in BCs, we designed a stickers and spacers model of phase-separating peptides interspersed with cysteines. Through biophysical investigations, we learned that cysteines promote liquid-liquid phase separation in oxidizing conditions and perpetuate liquid condensates through disulfide crosslinks, which can be reversibly tuned with redox chemistry. By varying the composition of cysteines, subtle but distinct changes in the viscoelastic behavior of the condensates were observed. Empirically, we conclude that cysteines are neither stickers nor spacers but function as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the role of cysteines in protein phase behavior and the potential to develop tunable, redox-sensitive viscoelastic materials.

biophysics↗

bioRxiv2024

Chemically-informed coarse-graining of electrostatic forces in charge-richbiomolecular condensates

Biomolecular condensates composed of highly charged biomolecules like DNA, RNA, chromatin, and nucleic-acid binding proteins are ubiquitous in the cell nucleus. The biophysical properties of these charge-rich condensates are largely regulated by electrostatic interactions. Residue-resolution coarse-grained models that describe solvent and ions implicitly are widely used to gain mechanistic insights into the biophysical properties of condensates, offering transferability, computational efficiency, and accurate predictions for many systems. However, their predictive accuracy diminishes for charge-rich condensates due to the implicit treatment of solvent and ions. Here, we present the Mpipi-Recharged model, a residue-resolution coarse-grained model that improves the description of charge effects in biomolecular condensates containing disordered proteins, multi-domain proteins, and/or disordered RNAs. Mpipi-Recharged maintains the computational efficiency of its predecessor--the Mpipi model--by still treating solvent and ions implicitly, but improves its accuracy by incorporating a pair-specific asymmetric electrostatic potential informed by atomistic simulations in explicit solvent and ions. We show that such asymmetric coarse-graining of electrostatic forces is needed to recapitulate the stronger mean-field impact of associative interactions between opposite-charge pairs over the repulsion among equally charged pairs revealed by our atomistic simulations. Mpipi-Recharged shows excellent agreement with the experimental phase behavior of highly charged systems, capturing subtle effects challenging to model without explicit solvation, such as the impact of charge blockiness, stoichiometry changes, and salt concentration variation. By offering improved predictions for charge-rich biomolecular condensates, Mpipi-Recharged extends the computational tools available to investigate the physicochemical mechanisms regulating biomolecular condensates.

biophysics↗

bioRxiv2024

Multiscale Responsive Kinetic Modeling: Quantifying Biomolecular Reaction Flux under Varying Electrochemical Conditions

Attaining a complete thermodynamic and kinetic characterization for processes involving multiple interconnected rare-event transitions remains a central challenge in molecular biophysics. This challenge is amplified when the process must be understood under a range of reaction conditions. Herein, we present a condition-responsive kinetic modeling framework that can combine the strengths of bottom-up rate quantification from multiscale simulations with top-down solution refinement using experimental data. Although this framework can be applied to any process, we demonstrate its use for electrochemically driven transport through channels and transporters. Using the Cl- /H+ antiporter ClC-ec1 as a model system, we show how robust and predictive kinetic solutions can be obtained when the solution space is grounded by thermodynamic constraints, seeded through multiscale rate quantification, and further refined with experimental data, such as electrophysiology assays. Turning to the Shaker K+ channel, we demonstrate that robust solutions and biophysical insights can also be obtained with sufficient experimental data. This multi-pathway method proves capable of identifying single-pathway dominant mechanisms but also highlights that competing and off-pathway flux is still essential to replicate experimental findings and to describe concentration-dependent channel rectification.

biophysics↗

bioRxiv2024

Caveolin assemblies displace one bilayer leaflet to organize and bend membranes

Caveolin is a monotopic integral membrane protein, widely expressed in metazoa and responsible for constructing enigmatic membrane invaginations known as caveolae. Recently, the high-resolution structure of a purified human caveolin assembly, the CAV1-8S complex, revealed a unique organization of 11 protomers arranged in a tightly packed, radially symmetric spiral disc. One face and the outer rim of this disc are hydrophobic, suggesting that the complex incorporates into membranes by displacing hundreds of lipids from one leaflet. The feasibility of this unique molecular architecture and its biophysical and functional consequences are currently unknown. Using Langmuir film balance measurements, we find that CAV1-8S is highly surface active, intercalating into lipid monolayers of various compositions. CAV1-8S can also incorporate into preformed bilayers, but only upon removal of phospholipids from the outer-facing leaflet. Atomistic and coarse-grained simulations of biomimetic bilayers support this leaflet replacement model and also reveal that CAV1-8S accumulates 40-70 cholesterol molecules into a disordered monolayer between the complex and its distal lipid leaflet. We find that CAV1-8S preferentially associates with positively curved membrane surfaces due to its influence on the conformations of distal leaflet lipids, and that these effects laterally sort lipids. Large-scale simulations of multiple caveolin assemblies confirmed their association with large, positively curved membrane morphologies consistent with the shape of caveolae. Further, association with curved membranes regulates the exposure of caveolin residues implicated in protein-protein interactions. Altogether, the unique structure of CAV1-8S imparts unusual modes of membrane interaction with implications for membrane organization, morphology, and physiology. STATEMENT OF SIGNIFICANCECaveolae are membrane invaginations heavily implicated in cellular physiology and disease; however, how their unique shape and function are produced remains enigmatic. Here, following on recent characterization of the unusual structure of the CAV1-8S oligomer, we examine the molecular details of its interactions with its surrounding lipid membrane using simulations and reconstitution experiments. We describe a novel mode of membrane interaction-which we term leaflet replacement-for the CAV1-8S complex that has not previously been observed for any other protein. The biophysical consequences of this unique molecular organization provide mechanistic insights into the functions and organization of caveolae in cells.

biophysics↗

bioRxiv2024

Maltodextrin Transport in the Extremely Thermophilic, Lignocellulose Degrading Bacterium Anaerocellum bescii (f. Caldicellulosiruptor bescii)

Sugar transport into microbial cells is a critical, yet understudied step in the conversion of lignocellulosic biomass to metabolic products. Anaerocellum bescii (formerly Caldicellulosiruptor bescii) is an extremely thermophilic, anaerobic bacterium that readily degrades the cellulose and hemicellulose components of lignocellulosic biomass into a diversity of oligosaccharide substrates. Despite significant understanding of how this microorganism degrades lignocellulose, the mechanisms underlying its highly efficient transport of the resulting oligosaccharides into the cell are comparatively underexplored. Here, we identify and characterize the ATP-Binding Cassette (ABC) transporters in A. bescii governing maltodextrin transport. Utilizing past transcriptomic studies on Anaerocellum and Caldicellulosiruptor species, we identify two maltodextrin transporters in A. bescii and express and purify their substrate-binding proteins (Athe_2310 and Athe_2574) for characterization. Using differential scanning calorimetry and isothermal titration calorimetry, we show that Athe_2310 strongly interacts with shorter maltodextrins such as maltose and trehalose with dissociation constants in the micromolar range, while Athe_2574 binds longer maltodextrins, with dissociation constants in the sub-micro molar range. Using a sequence-structure-function comparison approach combined with molecular modeling we provide context for the specificity of each of these substrate-binding proteins. We propose that A. bescii utilizes orthogonal ABC transporters to uptake malto-oligosaccharides of different lengths to maximize transport efficiency. ImportanceHere, we reveal the biophysical and structural basis for oligosaccharide transport by two maltodextrin ABC transporters in A. bescii. This is the first biophysical characterization of carbohydrate uptake in this organism and establishes a workflow for characterizing other oligosaccharide transporters in A. bescii and similar lignocellulosic thermophiles of interest for lignocellulosic bioprocessing. By deciphering the mechanisms underlying high affinity sugar uptake in A. bescii, we shed light on an underexplored step between extracellular lignocellulose degradation and intracellular conversion of sugars to metabolic products. This understanding will expand opportunities for harnessing sugar transport in thermophiles to reshape lignocellulose bioprocessing as part of a renewable bioeconomy.

biophysics↗

bioRxiv2024

Nanoscale curvature of the plasma membrane regulates mechanoadaptation through nuclear deformation and rupture

Nuclear translocation of the transcription regulatory proteins YAP and TAZ is a critical readout of cellular mechanotransduction. Recent experiments have demonstrated that cells on substrates with well-defined nanotopographies demonstrate mechanoadaptation through a multitude of effects - increased integrin endocytosis as a function of nanopillar curvature, increased local actin assembly on nanopillars but decreased global cytoskeletal stiffness, and enhanced nuclear deformation. How do cells respond to local nanotopo-graphical cues and integrate their responses across multiple length scales? This question is addressed using a biophysical model that incorporates plasma membrane (PM) curvature-dependent endocytosis, PM curvature-sensitive actin assembly, and stretch-induced opening of nuclear pore complexes (NPCs) in the nuclear envelope (NE). This model recapitulates lower levels of global cytoskeletal assembly on nanopillar substrates, which can be partially compensated for by local actin assembly and NE indentation, leading to enhanced YAP/TAZ transport through stretched NPCs. Using cell shapes informed by electron micrographs and fluorescence images, the model predicts lamin A and F-actin localization around nanopillars, in good agreement with experimental measurements. Finally, simulations predict nuclear accumulation of YAP/TAZ following rupture of the NE and this is validated by experiments. Overall, this study indicates that nanotopography tunes mechanoadaptation through both positive and negative feedback on mechanotransduction. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/619165v2_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@ec6d5aorg.highwire.dtl.DTLVardef@1ff6ec2org.highwire.dtl.DTLVardef@c09f46org.highwire.dtl.DTLVardef@18df50_HPS_FORMAT_FIGEXP M_FIG C_FIG This study uses experiments and biophysical modeling to examine the response and adaptation of cells to nanoscale topography of surfaces. It is shown that cytoskeletal assembly and nuclear localization of transcription regulatory factors such as YAP/TAZ can be tuned by nanoscale membrane curvature and nuclear deformation and rupture due to substrate nanotopography.

biophysics↗

bioRxiv2024

DeFrND: detergent-free reconstitution into native nanodiscs with designer membrane scaffold peptides

Membrane scaffold proteins-based nanodiscs (NDs) have facilitated unprecedented structural and biophysical analysis of membrane proteins in a near-native lipid environment. However, successful reconstitution of membrane proteins in NDs requires prior solubilization and purification in detergents, which may impact their physiological structure and function. Furthermore, the detergent-mediated reconstitution of NDs is unlikely to recapitulate the precise composition or asymmetry of native membranes. To circumvent this fundamental limitation of traditional ND technology, we herein describe the development of membrane-solubilizing peptides to directly extract membrane proteins from native cell membranes into nanoscale discoids. By systematically protein engineering and screening, we created a new class of chemically modified Apolipoprotein-A1 mimetic peptides to enable the formation of detergent-free NDs (DeFrNDs) with high efficiency. NDs generated with these engineered membrane scaffold peptides are suitable for obtaining high-resolution structures using single-particle cryo-EM with native lipids. To further highlight the versatility of DeFrNDs, we directly extract a sampling of membrane signaling proteins with their surrounding native membranes for biochemical and biophysical interrogations.

biophysics↗

bioRxiv2024

Conformational plasticity of mitochondrial VDAC2 controls the kinetics of its interaction with cytosolic proteins

The Voltage Dependent Anion Channel (VDAC) is the major conduit of water-soluble metabolites and small ions into and out of the mitochondria. In mammals, VDAC exists in three isoforms, VDAC1, VDAC2, and VDAC3, each characterized by distinct tissue-dependent distribution and physiological role. VDAC2 is the most notable among the three isoforms because its knockout results in embryonic lethality and regulates the BAK/BAX-dependent apoptosis pathways. Yet, understanding of the biophysical underpinnings of VDAC2 functions remains limited. In this study, we reevaluate VDAC2s properties, utilizing recombinant human VDAC2 WT and its three mutants - cysteine-less VDAC2, VDAC2 with truncated first 11 residues, and E84A - to explore the biophysical basis that distinguishes VDAC2 from the other isoforms using single-molecule electrophysiology. We found that contrary to VDAC1 and VDAC3, which are characterized by a unique open state, VDAC2 displays dynamic switching between a few high-conductive anion-selective substates. We employed -synuclein (Syn) - a known potent cytosolic regulator of VDAC1 and VDAC3 - as a sensitive molecular probe to show that it induces characteristic blockage events in all open substates of VDAC2 but with up to ten-fold different on-rates and blockage times. A substate with higher conductance always corresponds to a higher on-rate of the Syn-VDAC2 interaction but proportionally lower blockage times. This gives the same equilibrium constant for all substates, thus resulting in the same affinity of the Syn-VDAC2 interaction. The pronounced difference is limited to the kinetic parameters, suggesting that once the Syn molecule is captured, its physical state and free energy are the same for all substates. These striking results imply that the Syn molecule senses the dynamic structural variations within the channel prior to its final capture by the pore. We propose that the discovered conformational flexibility may allow VDAC2 to recognize a larger number of binding partners, thus explaining the physiological significance of this isoform, namely, its ability to adapt to mitochondrial metabolic conditions in cells dynamically.

biophysics↗

bioRxiv2024

Sensing the shape of a surface by intracellular filaments

Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as Escherichia coli by directing cell-wall synthesis according to local curvature cues. However, the basis of MreBs curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of [~]50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization, and suggest new design principles for synthetic shape sensing nanomaterials. Significance statementThe protein MreB, a homolog of eukaryotic actin, regulates the shape of bacteria like Escherichia coli by guiding new cell-wall insertion based on local curvature cues. However, the mechanism by which a nanometer-scale MreB filament "senses" the micron-scale curvature of the cell wall has remained a mystery. We introduce a biophysical model of the energetics of twisted and bent filaments bound to curved surfaces, which predicts that localization of filaments like MreB is sensitive to both mean and Gaussian curvature. The model captures experimentally measured curvature enrichment patterns and explains how MreB naturally localizes to saddle-shaped regions without energy-consuming processes. Beyond cell shape regulation, our work suggests design principles for synthetic systems that can sense and respond to surface shape.

biophysics↗

bioRxiv2024

Glycocalyx-induced formation of membrane tubes

Tubular membrane structures are ubiquitous in cells and in the membranes of intracellular organelles such as the Golgi complex and the endoplasmic reticulum. Tubulation plays essential roles in numerous biological processes, including filopodia growth, trafficking, ion transport, and cellular motility. Understanding the fundamental mechanism of the formation of membrane tubes is thus an important problem in the fields of biology and biophysics. Though extensive studies have shown that tubes can be formed due to localized forces acting on the membrane or by the curvature induced by membrane-bound proteins, little is known about how membrane tubes are induced by glycocalyx, a sugar-rich layer at the cell surface. In this work, we develop a biophysical model that combines polymer physics theory and the Canham-Helfrich membrane theory to investigate how the glycocalyx generates cylindrical tubular protrusions on the cell membrane. Our results show that the glycocalyx alone can induce the formation of tubular membrane structures. This tube formation involves a first-order shape transition without any externally applied force or other curvature-inducing mechanisms. We also find that critical values of glycocalyx grafting density and glycopolymer length are needed to induce the formation of tubular structures. The presence of vertical actin force, line tension, and spontaneous curvature reduces the critical grafting density and length of polymer that triggers the formation of membrane tube, which suggests that the glycocalyx makes tube formation energetically more favorable when combined with an actin force, line tension, and spontaneous curvature. Significance StatementIn many cells, the existence of glycocalyx, a thick layer of polymer meshwork comprising proteins and complex sugar chains coating the outside of the cell membrane, regulates the formation of membrane tubes. Here, we propose a theoretical model that combines polymer physics theory and the Canham-Helfrich membrane theory to study the formation of cylindrical tubular protrusions induced by the glycocalyx. Our findings indicate that glycocalyx plays an important role in the formation of membrane tubes. We find that there exists critical grafting density and length of polymer that triggers the formation of membrane tubes, and the glycocalyx-induced tube formation is facilitated when combined with actin forces, line tension, and spontaneous curvature. Our theoretical model has implications for understanding how biological membranes may form tubular structures.

biophysics↗

bioRxiv2024

Unbiased learning of protein conformational representation via unsupervised random forest

Accurate data representation is paramount in biophysics to capture the functionally relevant motions of biomolecules. Traditional feature selection methods, while effective, often rely on labeled data based on prior knowledge and user-supervision, limiting their applicability to novel systems. Here, we present unsupervised random forest (URF), a self-supervised adaptation of traditional random forests that identifies functionally critical features of biomolecules without requiring prior labels. By devising a memory-efficient implementation, we first demonstrate URFs capability to learn important sets of inter-residue features of a protein and subsequently to resolve its complex conformational landscape, performing at par or surpassing its traditional supervised counterpart and 15 other leading baseline methods. Crucially, URF is supplemented by an internal metric, the learning coefficient, which automates the process of hyper-parameter optimization, making the method robust and user-friendly. URFs remarkable ability to learn important protein features in an unbiased fashion was validated against 10 independent protein systems including both both folded and intrinsically disordered states. In particular, benchmarking investigations showed that the protein representations identified by URF are functionally meaningful in comparison to current state-of-the-art deep learning methods. As an application, we show that URF can be seamlessly integrated with downstream analyses pipeline such as Markov state models to attain better resolved outputs. The investigation presented here establishes URF as a leading tool for unsupervised representation learning in protein biophysics.

biophysics↗

bioRxiv2024

Agent-based modeling reveals impacts of cell adhesion and matrix remodeling on cancer collective cell migration phenotypes

Understanding the phenotypic transitions of cancer cells is crucial for elucidating tumor progression mechanisms, particularly the transition from a non-invasive spheroid phenotype to an invasive network phenotype. We developed an agent-based model (ABM) using Compucell3D, an open-source biological simulation software, to investigate how varying biophysical and biochemical parameters influence emerging properties of cellular communities, including cell growth, division, and migration. Our focus was on cell-cell contact adhesion and matrix remodeling effects on cancer cell migration. We simplified enzymatic remodeling of the extracellular matrix and the subsequent enhancements to cellular chemotaxis or durotaxis as a combined effect of localized cellular secretion of a chemoattractant. By varying the chemoattractant secretion rate and contact adhesion energy, we simulated their effects on cellular behavior and driving the transition from a spheroid phenotype to a network phenotype. The model serves as a digital twin for 3D cancer cell culture, simulating cancer cell growth, division, and invasion over 1 week, validated against published data. The simulations track the emergent morphological and collective phenotype changes using key metrics such as cell circularity and invasion. Our findings indicate that increased chemoattractant secretion enhances the invasiveness of the collective cells, promoting the transition to a network phenotype. Additionally, changing cell-cell contact energy from a strong cell-cell adhesion to a weak cell-cell adhesion affects the compactness of the spheroids, resulting in lower circularity and increased collective cell invasion. Our work advances the understanding of tumor progression by providing insights into the biophysical mechanisms behind invasive cancer cell phenotypic transitions.

biophysics↗

bioRxiv2025

Exploring the kinetics and mechanism of phase separation in ternary lipid mixtures containing APP C99 using atomistic vs coarse-grained MD simulations

The phase separation of lipid bilayers, composed of mixtures of saturated and unsaturated lipids and cholesterol, is a topic of fundamental importance in membrane biophysics and cell biology. The formation of lipid domains, including liquid-disordered domains enriched in unsaturated lipids and liquid-ordered domains enriched in saturated lipids and cholesterol is believed to be essential to the function of many membrane proteins. Experiment, theory, and simulation have been used to develop a general understanding of the thermodynamic driving forces underlying phase separation in ternary and quaternary lipid mixtures. However, the kinetics of early events in lipid phase separation in the presence of transmembrane proteins remain relatively understudied. Using large-scale all-atom and coarse-grained simulations, we explore the kinetics and phase separation of ternary lipid mixtures of saturated lipid, unsaturated lipid, and cholesterol. Order parameters employed in the Cahn-Hilliard theory provide insight into the kinetics and mechanism of lipid phase separation. We observe three distinct time regimes in the phase separation process: a shorter time exponential phase followed by a power law phase followed by a longer time plateau phase. Comparison of lipid, protein and lipid-protein dynamics between all-atom and coarse-grained models identifies both quantitative and qualitative differences and similarities in the phase separation kinetics. Moreover, timescaling of dynamics of AA and CG simulation yields a similar kinetic mechanism of phase separation. The findings of this study elucidate fundamental aspects of membrane biophysics and the ongoing efforts to define the role of lipid rafts in the structure and function of cellular membrane.

biophysics↗

bioRxiv2025

Lipopolysaccharides nanodiscs, a biomimetic platform to study bacterial surface.

Lipopolysaccharides (LPS) are essential components of the outer membranes of Gram-negative bacteria, playing a crucial role in antimicrobial resistance, virulence, and the hosts immune response. Self-assembled particles displaying LPS are essential for biophysical studies addressing the behavior of bacterial surfaces under specific biomimetic conditions. Styrene-maleic acid (SMA) copolymers were employed to form LPS nanodiscs, either from extracted LPS or directly from purified outer membranes. These nanodiscs, derived from pathogenic O157:H7 or laboratory E. coli strains, are well-defined in size and yield high-resolution nuclear magnetic resonance (NMR) spectra. They have been successfully used to investigate molecular recognition by a human C-type Lectin Receptor (CLR) of the immune system and interaction with polymyxin antibiotics using various biophysical methods. This study high-lights the significance of LPS nanodiscs as bacterial surface mimetics and opens promising avenues for further research into LPS structure and interactions. The ability to generate well-defined LPS nanodiscs offers a powerful tool for studying the molecular mechanisms underlying bacterial pathogenesis and immune response.

biophysics↗