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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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32 records · Page 2Linked to original sources

bioRxiv2026

The Gordian Knot Enhances Ubiquitin Binding in UCH-L1

UCH-L1 is a monomeric deubiquitinating enzyme whose native structure embeds a shallow $5_2$ knot located near the N-terminus, placing the knotted topology in direct proximity to both the substrate-binding pocket and the catalytic site. While our previous work established that N-terminal integrity is critical for catalytic activity, the energetic cost of unknotting and its structural consequences remained unquantified. Here, we combine steered molecular dynamics with an umbrella sampling scheme to generate topologically modified variants of UCH-L1 and, for the first time, reconstruct the free-energy profile of UCH-L1 unknotting. The potential of mean force reveals a steep energetic barrier to knot disruption, consistent with knotting being a late, rate-limiting folding step that is effectively locked in once the native structure is established. Long unbiased MD simulations of fully unknotted variants in both apo and holo states show that knot removal increases local flexibility at the N-terminus without inducing significant global structural destabilization. Binding energy calculations indicate that the unknotted variant binds to ubiquitin less tightly than the wild-type ($\sim$-62~vs~$\sim$-76~kcal/mol), suggesting that topological integrity contributes to substrate affinity. Together, these results show that the $5_2$ knot in UCH-L1 is not a passive structural feature but a functional element that fine-tunes folding kinetics and contributes to substrate binding efficiency.

biophysics↗

bioRxiv2026

Quantifying sprint force-velocity elasticity: implications for individualized training decisions

This study aimed to (1) develop an elasticity framework for the sprint force-velocity (F-V) relationship and (2) examine how maximal force (F_{0}), maximal velocity (v_{0}), and sprint distance modulate the four derived elasticity metrics, and (3) explore these elasticity metrics' interrelation. After modelling the F-V relationship differential equation, four elasticity metrics were defined as force elasticity (F_{e}), the elasticity of sprint time to F_{0}; velocity elasticity (v_{e}), the elasticity of sprint time to v_{0}; the force-velocity elasticity norm {(\mathrm{F}-\mathrm{V}}_{\mathrm{EN}}=\sqrt{F_{e}^{2}+v_{e}^{2}}), capturing the combined sprint time sensitivity to proportional changes in F_{0} and v_{0}; and the force-velocity elasticity ratio {(\mathrm{F}-\mathrm{V}}_{\mathrm{ER}}=F_{e}{\div v}_{e}), indicating which variable dominates the sprint time response. Model simulations showed that F_{e} decreased with rising F_{0} and increased with rising v_{0}, while v_{e} showed the opposite pattern. With increasing sprint distance, F_{e} decreased and v_{e} increased. Given its negligible effect on sprint time, ignoring air resistance yields a conservation law (2F_{e}+v_{e}\equiv 1), indicating that a gain in one elasticity metric necessarily diminishes the other in a fixed proportion. This framework also identifies a valley distance (d_{valley}) at {\mathrm{F}-\mathrm{V}}_{\mathrm{ER}}=2, where {\mathrm{F}-\mathrm{V}}_{\mathrm{EN}} is minimized (\sqrt{0.2}) and sprint time is least responsive to changes in F-V relationship variables. Empirical data confirmed that the two theoretical laws still hold approximately when air resistance is considered. By linking changes in F_{0} and v_{0} to sprint time across different distances, the elasticity framework provides a quantitative basis for estimating the theoretical sprint time response to documented changes in F-V relationship variables.

biophysics↗

bioRxiv2026

Local mechanical heterogeneity drives epidermal cell delamination

Delamination within stratified epithelia like the skin epidermis describes the detachment and upward motion of cells originating from the basal layer. Despite its fundamental importance for tissue development, homeostatic regeneration and repair, the mechanisms that drive delamination remain a longstanding open question. Upward motion follows cell shape changes, which are inherently driven by physical forces, but their role is elusive. Here, we investigate delamination in stratifying keratinocytes by combining imaging, force measurements and theoretical modeling. We identify a local change in force balance between differentiating cells and their environment as the key step initiating delamination. Within a homogeneous cell layer with apically polarized contractility, differentiation leads to actomyosin remodeling, redistributing cellular force exertion to the basal side. Such mechanical heterogeneity then results in differentiating cells experiencing and inward basal and outward apical forces that manifest in the formation of a +1 force defect and promote shape changes culminating in upward motion. Simultaneously, delaminating cells actively pull on their underlying neighbors, generating convergent tissue flows which close the basal layer below. Together, we propose a general physical description of delamination initiation, which may act across various multilayered epithelia.

biophysics↗

bioRxiv2026

Stochastic Biophysics of Cellular Radiosensitivity: From Molecular Noise and Repair Kinetics to Evolutionary Demographics

Radiation-induced DNA double-strand breaks (DSBs) drive cellular mortality, mutagenesis, and severe evolutionary bottlenecks. While classical phenomenological models, such as the Linear-Quadratic (LQ) framework, reliably predict macroscopic population survival, they obscure the intrinsic single-cell stochasticity that governs critical rare events like tumor recurrence or the emergence of radioresistant persisters. To bridge this divide, we develop a mathematically exact stochastic differential equation (SDE) framework that models continuous DSB induction and repair as a Feller square-root process. By deriving exact closed-form expressions for the foci moments, we establish a highly efficient Maximum Likelihood Estimation (MLE) pipeline that circumvents computationally exhaustive Monte Carlo simulations, allowing the direct extraction of deterministic repair velocities and intrinsic molecular noise from empirical single-cell $\gamma$-H2AX data. Integrating this kinetic model with a cumulative damage hazard via the Feynman-Kac formalism, our framework seamlessly recovers the classic macroscopic LQ survival topology from microscopic first principles. Furthermore, systematic sensitivity analysis uncovers a fundamental evolutionary duality: while initial physical damage operates additively, ultimate cellular fate is driven by a nonlinear survival response governed by the trade-off between the damage hazard rate and intrinsic molecular noise strength. Crucially, we demonstrate that this molecular noise inherently enhances population survival. Governed by Jensen's inequality, stochastic variance acts as a non-genetic bet-hedging mechanism that buffers the population by favoring cells with transiently low damage loads. Ultimately, this exact stochastic framework bridges microscopic biophysics and macroscopic demographics, offering deep mechanistic insights into the evolutionary roots of radioresistance.

biophysics↗

bioRxiv2026

Polarized neutrons for the study of individual and collective fast dynamics in proteins

Neutron scattering is a powerful technique to investigate atomic structures and molecular dynamics of proteins at the nano-scale. When it comes to dynamics, incoherent and coherent scattering respectively provide information on the single and collective dynamics of nuclei. In proteins, hydrogen has the highest incoherent cross-section, and it is common practice to overlook the contribution of coherent terms stemming from all nuclei. However, the fast collective dynamics of heavier nuclei could also be studied if coherent scattering and incoherent scattering were experimentally separated. The recent advent of polarized neutron spectroscopy with sufficient flux and energy resolution has made it possible, and opens new perspectives to investigate the relative importance of coherent scattering and the information it provides on biological samples. The present study reports on the use of polarized quasi-elastic neutron scattering (QENS) and the application of a minimalistic model adapted to both individual and collective dynamics. Using a perdeuterated green fluorescent protein as a model globular protein, the study provides an interpretation of the dynamical parameters obtained with QENS, and a comparative study of the Elastic Coherent and Incoherent Scattering Factor. Based on both experiments and calculations, we discuss the relative importance of distinct and self components of coherent scattering, which is often wrongly assumed to be representative of collective dynamics only. The results highlight the current impediments rendering complicated a straightforward analysis of fast collective dynamics in hydrated protein samples.

biophysics↗

bioRxiv2026

Multivalent Adhesive Probe Atomic Force Microscopy (MAPA) for accessing dispersive adhesion of cells and biosurfaces.

Adhesion of cells is the key factor determining functioning of multicellular organisms. Viscoelastic properties of cells can be studied by multiple methods. However, attractiveness of cells or extracellular matrix without the elastic component (dispersive adhesion) is not accessible. We present an extension of force spectrometry technology: the Multivalent Adhesive Probe Atomic Force Microscopy (MAPA) that delivers dispersive adhesion maps of live cells and biosurfaces, and identifies differences unresolved by viscoelastic probing.

biophysics↗

bioRxiv2026

3D Printed X-ray Compatible Microfluidics for Online Characterization of Hexosomes: A Synchrotron SAXS-on-Chip Study with Molecular Dynamics Insights

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

biophysics↗

bioRxiv2026

THE ROLE OF LIQUID CRYSTAL ORDERING IN THE STRUCTURAL ORGANIZATION OF DNA IN BACTERIA.

This paper presents and critically reviews the results of original and some literature based experimental studies conducted by the authors last years on the structural organization of DNA in dormant (starvation stress), anabiotic dormant (4 HR treatment) E. coli cells, as well as the K12 {Delta}dps strain, which lacks the Dps protein (Dps null E. coli). The experimental data includes small-angle synchrotron radiation diffraction (SAXS) and transmission electron microscopy (TEM) data. Synchrotron radiation diffraction experiments on K12{Delta}dps cells allowed us to conclude that peaks at 44.3, 22.1, and 14.8 angstrom resolutions are associated exclusively with ordered DNA organization. Peaks at 44.3, 22.1, and 14.8 angstrom resolutions are also observed for samples of dormant (starvation stress) cells and anabiotically dormant cells. Therefore, this ordered DNA organization also applies to samples of dormant and anabiotically dormant cells. A model is proposed that considers the ordered DNA organization in the cell as a cholesteric liquid crystal. The powder diffraction pattern calculated based on this model is compared with experimental small angle X ray scattering (SAXS) data obtained on Dps-null cell samples. The model completely reproduces the key features of the experimental diffraction pattern from Dps-null cell samples. Accordingly, the cholesteric liquid crystal model corresponds to DNA packaging in dormant and anabiotically dormant cells. Cholesteric liquid crystal ordering should be further considered in all models of cellular DNA packaging. To address the question of which structural organization of DNA predominates in the cell: the cholesteric liquid crystal or nanocrystalline or whether they coexist and fully manifest themselves under different external conditions, it is necessary to utilize the latest methodological advances in structural analysis.

biophysics↗

bioRxiv2026

Topological Closure Drives Structural Stabilization and Fast Cooperative Dynamics in Crowded Circular Polysomes

In linear polysomes, excluded-volume interactions among ribosomes can induce dimensional reduction of mRNA. Yet linear architectures allow steric stress to relax at open ends-- limiting how strongly crowding can remodel the mRNA's structure and dynamics. Using coarse-grained molecular-dynamics simulations, we compare circular and linear polysomes over a range of ribosome densities. Circular closure selects a predominantly quasi-planar global conformational ensemble, as indicated by a shape dimensionality dshape {approx} 2 over a range of ribosome densities. Crucially, circular topology and ribosome crowding act cooperatively to suppress structural fluctuations. While closure alone or linear crowding reduces relative global size fluctuations ({Delta}Rg/Rg) only to {approx} 0.16, their combined effect drives this fluctuation down to {approx} 0.07. Within this stabilized architecture, increasing ribosome density drives a distinct in-plane reorganization: the ring becomes more isotropic, global size fluctuations are strongly suppressed, and the scaling exponent increases toward {nu} [~=] 0.74 - 0.77, consistent with two-dimensional self-avoiding walk-like value over the accessible finite-size window, 1000 [≤] N [≤] 4969. Closure shortens the radius-of-gyration decorrelation time of circular polysomes by 40-fold relative to matched linear systems, reflecting the topological elimination of free ends. Within this closureselected ensemble, ribosome crowding further reduces the decorrelation time by up to 20% at the highest density. A fluctuation-informed crossover model links the density dependence of the global scaling exponent to inter-ribosomal subchain statistics. These results distinguish the geometric role of circular closure from the density-dependent steric response that it enables, revealing a confined yet dynamically responsive conformational regime for circular polysomes.

biophysics↗

bioRxiv2026

Molecular basis of AMPA receptor labeling by ligand-directed acyl imidazole chemistry in living neurons

Rational design of covalent protein-labeling reagents in complex biological environments requires a molecular-level understanding of how the protein microenvironment governs chemical reactivity; yet, such mechanistic details remain inaccessible to experimental methods alone. In living neurons, Ligand-Directed Acyl Imidazole (LDAI) chemistry has been used to label AMPA receptors as a traceless, affinity-based protein labeling method. Although LDAI labeling reagents have been optimized in the lab, the atomic details of their interactions with the protein and the underlying mechanism remain elusive. In this work, we combined Quantum Mechanical (QM) calculations and molecular dynamics (MD) simulations to propose a detailed reaction mechanism for AMPAR labeling by LDAI reagents and to clarify how the protein microenvironment governs reactivity. Although Lys residues are usually protonated at physiological pH and therefore less nucleophilic in water, our QM results show that Lys labeling is energetically more favorable than competing reactions with Ser or water. MD simulations reveal that PFQX ---the LDAI reagent precursor--- binds dynamically to the GluA2 AMPAR as an antagonist, inducing conformational changes that reshape the local environment of the acyl imidazole (AI) warhead, underscoring that ligand identity strongly affects labeling outcomes. We also identified intra and intermolecular hydrogen bond networks that may contribute to further immobilize and pre-organize the LDAI reagent. Moreover, the probe's chemical nature shapes its interactions with the Ligand Binding Domain (LBD), offering a plausible rationale for the previously experimentally observed ligand-dependent fluorescent response. Taken together, our results establish design principles for exploiting the reagent geometry and binding pocket hydrogen-bonding networks for the rational design of LDAI reagents.

biophysics↗

bioRxiv2026

Low-Density Lipoprotein Modulates Plasma Fibrin Network Architecture and Impairs Fibrinolysis

Low-density lipoprotein (LDL) is a major atherogenic lipoprotein, yet its potential to directly modify the fibrin scaffold of blood clots is incompletely understood. Here, we investigated how LDL alters plasma fibrin network architecture and internal fibrinolysis across defined fibrinogen/thrombin conditions. Pooled normal human plasma was supplemented with LDL and clotted with controlled concentrations of fibrinogen and thrombin. Fibrin architecture was visualized by confocal microscopy and quantified by pore-size analysis; clot formation and lysis were monitored turbidimetrically in the presence of tissue plasminogen activator (tPA). Increasing LDL produced a pronounced reduction in fibrin-network pore size across the tested fibrinogen/thrombin conditions. The LDL dependence of pore diameter was well described by a power-law relationship, D_pore=(6.54 +/- 0.11)[LDL]^(-0.12 +/- 0.02) , (R^2 = 0.90), with a significant negative LDL exponent (p = 4 x 10^5). Increasing LDL also prolonged clot lysis time and altered turbidity kinetics. These findings extend epidemiologic and clinical associations between ApoB-containing lipoproteins and hypofibrinolytic clot phenotypes by demonstrating, in a controlled plasma system, that LDL itself can modify fibrin network architecture and fibrinolytic susceptibility. The results support a structure-function role for LDL within the fibrin biomaterial and motivate direct tests of LDL incorporation, protofibril packing, fibrinolytic-protein binding, and single-fiber mechanics.

biophysics↗

bioRxiv2026

Resolving Heterogeneous Mechanical Domains via Physics-Aware Deep Clustering of Single-Molecule Force Spectroscopy Data

Many biological processes rely on mechanical forces, with protein molecules acting as key mediators. Understanding how proteins respond to mechanical stress is essential for conditions including cardiomyopathy and muscular dystrophy. Natural proteins such as dystrophin and utrophin are composed of heterogeneous folding domains with distinct mechanical properties; deciphering domain-level behavior provides insights into disease mechanisms and informs therapeutic strategies. Single-molecule force spectroscopy (SMFS) enables probing the mechanical properties of entire proteins, yet current approaches struggle to identify heterogeneous folding domains, particularly without prior knowledge. Here, we present the first automated framework to identify heterogeneous folding domains in SMFS data, applying both existing clustering methods and a novel physics-aware deep clustering architecture, LatentUnfold. LatentUnfold learns complementary latent representations from force magnitude and the force-extension physical relationship through dual autoencoders, jointly optimized for clustering assignments. We apply our framework to experimental SMFS data collected from a synthetic two-domain protein (ddFLN4-Titin I27) as well as natural protein constructs of dystrophin and utrophin, with Monte Carlo simulated datasets serving as controlled validation. For the synthetic protein, we recover mechanical properties consistent with previously reported values for each domain. For the natural proteins, we uncover two mechanically distinct domain populations - corresponding to the N-terminal domain and spectrin-like repeats - with differences in both unfolding force and contour length increase, and reveal different unfolding order between them for the first time. This work enables domain-level biological inference, overcoming prior limitations that relied on averaging and overlooked heterogeneity, thus advancing the understanding of mechanical behavior in protein unfolding.

biophysics↗

bioRxiv2026

Trans-Allosteric Activation Releases Distinct Conformational Traps in Kinase Heterodimers

Protein kinases function as dynamic, mechanically coupled nodes, yet the conformational drivers of multimeric activation remain unclear. Here, we present AlloQuant, a computational suite that translates AlphaFold3 structural ensembles into quantitative metrics of kinase regulation, including internal network rigidity, metastable-state populations, and sub-angstrom conformational drivers. Applying AlloQuant to CDK1, we demonstrate that binding of the Cyclin B1 (CCNB1) cofactor mechanically decouples a hyper-rigid inactive kinase core, allowing activating phosphorylation (pT161) to subsequently re-impose localized tension on the catalytic machinery. Conversely, the C-terminal Src kinase (CSK) faces a distinct conformational trap. While nucleotide-free monomeric CSK spontaneously samples a pre-active geometry, ATP binding excludes the active C-In conformation in all but 1 of 225 models. We show that docking partner engagement overcomes this blockade. Autophosphorylation of SRC at the activation loop (Y419) redistributes SRC conformational states without altering bulk rigidity. This redistribution is structurally coupled to the conformational state of CSK via the regulatory spine, not the catalytic machinery. Rather than mechanically deforming CSK, SRC engagement acts by conformational selection, committing roughly a quarter of CSK molecules to a fully active state. Thus, trans-allosteric kinase activation operates by defining the accessible conformational landscape of the receiver kinase. That control is exerted through mechanical remodeling in cofactor-dependent complexes and through conformational selection in transient kinase-kinase heterodimers. These findings establish AlloQuant as a general framework for quantifying how a binding partner reshapes a kinase's conformational landscape, applicable across the kinome because it assigns landmarks by profile-HMM alignment.

biophysics↗

bioRxiv2026

Dehydration triggers anomalous subdiffusion in biomimetic cell membranes

Lipid diffusion plays a central role in shaping the structural organization of cell membranes, maintaining lipid homeostasis, and facilitating cellular transport and signaling. The lateral mobility of phospholipids in membranes depends heavily on their hydration state. Furthermore, the activation energy of diffusion increases in conditions of reduced membrane hydration, suggesting that the underlying diffusion mechanism changes upon dehydration. Using two variants of fluorescence correlation spectroscopy (point FCS and scanning FCS) and two membrane reporters, we demonstrate that mild dehydration of phase-separated biomimetic cell membranes alters the lipid diffusion mechanism, resulting in anomalous subdiffusion rather than free Brownian motion. Importantly, the anomalous diffusion parameter, , decreases significantly upon the initial reduction of the membrane hydration layer, and the effect is fully reversible upon rehydration. These observations strongly indicate the reversible shift in lipid diffusion mode rather than irreversible membrane damage. We propose that this anomalous subdiffusion is caused by the formation of temporarily immobile lipid pockets in the membrane upon dehydration. These results therefore provide important insights into the mechanism of lipid diffusion in membranes undergoing local and transient dehydration, which is an important intermediate step in various biological processes associated with membrane fusion, such as neurotransmission, fertilization, and viral entry.

biophysics↗