Biophysical Journal

Lipid membranes undergo liquid-liquid phase separation to form micron-scale domains. Several physical parameters affect this phase transition. For example, a decrease in temperature causes membranes to demix, as does an increase in hydrostatic pressure. However, measurements to determine how tension affects membrane phase separation have yielded conflicting results. Experiments that have applied osmotic pressure differences to a population of vesicles have reported an increase in the membrane's miscibility transition temperature. Conversely, experiments that have applied micropipette aspiration or substrate stretching to single membranes have reported a decrease. Here, we find that both osmotic pressure and micropipette aspiration can increase transition temperatures. We discuss how membrane pores and hidden areas in membranes present challenges to researchers seeking to quantitatively convert experimentally measured osmolarity differences into membrane tensions. We show that challenges of comparing data from different osmotic pressure experiments can be mitigated by renormalizing osmolarity differences, specifically by dividing by the exterior osmolarity. We discuss our results in the context of existing theoretical predictions and in light of four known effects of increasing tension on vesicle membranes: 1) a reduction in hidden area of tubes and aggregates, 2) a reduction in out-of-plane thermal fluctuations, 3) an increase in the area per lipid, and 4) the formation of pores. First, hidden area can explain why vesicles can sustain high osmolarity differences. Next, suppression of thermal fluctuations and increases in the area per lipid may account for shifts in miscibility transition temperatures. Finally, pores can explain time dependences. Overall, our results highlight the need for new theory and simulation that unify predictions of how transition temperatures vary over all four regimes of membrane tension.

In muscle cells, desmin intermediate filaments form a cytoskeletal network that maintains the structural integrity and mechanical coupling of myofibrils. Dominant missense mutations in desmin cause myopathies characterized by intracellular protein aggregation. To explore the process of aggregate formation, we investigate the assembly kinetics of wild-type desmin and four disease-associated variants (N342D, L345P, R350P, and R406W) using dual-wavelength stopped-flow spectroscopy, complemented by atomic force microscopy and molecular dynamics simulations. At low ionic strength, all proteins form uniform tetramers. Increasing the ionic strength initiates assembly byrapid lateral association of tetramers into unit-length filaments (ULFs), followed by longitudinal elongation and radial filament compaction. R406W forms ULFs with wild-type-like kinetics, whereas N342D, L345P, and R350P exhibit delayed lateral assembly. After short filaments have formed, all four mutants diverge from productive filament maturation, but through distinct pathways. Quantitative analysis of atomic force microscopy images together with kinetic modelling of the spectroscopic data shows that wild-type filaments elongate continuously, whereas R406W filaments progressively associate into fibrillar clusters and cease elongating. By contrast, N342D, L345P, and R350P rapidly collapse into globular complexes that subsequently coalesce into larger aggregates. Molecular dynamics simulations indicate that the mutations differentially destabilize coil 2, which leads to local structural perturbations and mutation-specific assembly defects. Together, these findings identify early filament maturation - when elongating ULF-derived filaments would normally undergo radial compaction to form stable, mature filaments - as a critical time point in desmin assembly. At this stage, pathogenic mutations redirect the internal reorganization of the filament from productive stabilization toward mutation-specific structural collapse and aggregation. SignificanceDominant mutations in the intermediate filament protein desmin cause myofibrillar myopathies characterized by protein aggregation and progressive muscle degeneration. Using in vitro filament-assembly experiments, we identify intrinsic assembly defects of disease-associated desmin mutants. Mutant proteins initially enter the normal assembly pathway, albeit with delayed kinetics. They subsequently fail at an early maturation step in which short filaments would normally reorganize to sustain productive elongation. Instead, mutant proteins form morphologically distinct aggregates. Similar structural abnormalities occur when mutant proteins co-assemble with wild-type desmin. Our findings identify the radial compaction phase during early longitudinal assembly as a critical checkpoint that determines whether desmin assembly yields functional intermediate filaments or is redirected into pathogenic aggregation pathways.

Dynamic conformational changes in intracellular domains fundamentally affect the function and pharmacology of many membrane proteins. For example, sodium influx through the cardiac voltage-gated sodium channel (NaV1.5) is rapidly terminated through conformational changes that result in pore closure, a transition known as inactivation. Inactivation involves NaV1.5 intracellular regions, particularly the DIII-DIV linker containing the IFM particle (Isoleucine-Phenylalanine-Methionine) and its dysfunction is a major cause of cardiac arrythmias. However, the conformational changes involved in inactivation and their modulation by auxiliary proteins and clinically used drugs remain incompletely characterized, partly because live-cell, site-specific labeling of intracellular regions with small fluorescent dyes remains challenging. Here, we combine live-cell protein semi-synthesis with voltage-clamp fluorometry (VCF), to track intracellular conformational dynamics of the cardiac sodium channel NaV1.5 and monitor their voltage dependence and kinetics in real time. We identify intracellular conformational changes involved in both fast and steady-state inactivation of NaV1.5 and show that both lidocaine and auxiliary proteins affect the kinetics of conformational changes of the DIII-DIV linker. Our work establishes the combination of protein semi-synthesis and voltage-clamp fluorometry as a powerful approach to dissect intracellular conformational changes in membrane proteins.

Membrane fusion is an essential process in cells that requires a balance of lipid composition to establish biophysical properties conducive to topology changes. During fusion, lipids of opposing membranes must invert and overcome energy barriers associated with forming highly curved stalk and pore intermediates. While theoretical work has modelled the effect of lipid intrinsic curvature on stalk formation, quantifying the relationship experimentally has proven to be a challenge due to the inability to vary lipid curvature without concomitantly changing other properties that affect fusion. Here we address this hurdle by using hydrostatic pressure to modulate lipid intrinsic curvature independently of chemical composition. Using high-pressure stopped-flow fluorimetry, we measured rates of calcium-mediated lipid mixing between populations of vesicles, a process that is strongly inhibited by pressure. We correlated mean lipid intrinsic curvature across pressure with lipid mixing rates by incorporating complementary small-angle x-ray scattering measurements for each individual lipid component. This analysis showed that lipid mixing rates, a proxy for hemifusion, across compositional and pressure regimes are determined by changes in lipid spontaneous curvature. Consistent with previous theoretical models, we find a linear relation between lipid intrinsic curvature and the hemifusion stalk formation energy, offering direct experimental support for the stalk hypothesis. Significance statementMembrane fusion proceeds through a hemifusion stalk intermediate whose formation energy depends on lipid intrinsic curvature, a central prediction of the stalk hypothesis that has lacked direct experimental support. Previous tests relied on changes in lipid composition that affect multiple membrane properties, confounding the contribution of curvature alone. Here we use hydrostatic pressure to tune lipid curvature independently of chemical composition and calibrate its effects with high-pressure SAXS. Hemifusion rates across three lipid compositions and four pressures collapse into a single exponential dependence on mean spontaneous curvature, yielding a linear relation between curvature and energy consistent with continuum elastic theory. This work quantifies how lipid composition tunes fusion kinetics, suggesting that small changes in lipid curvature may strongly affect fusogenicity.

Adherent cells must continuously adapt to rapid environmental fluctuations to preserve mechanical integrity. However, conventional theoretical frameworks predominantly rely on quasi-static assumptions, thereby limiting their ability to capture the transient dynamics of cellular responses to high-rate perturbations, such as acute osmotic shocks. To address this limitation, we developed a biophysical model incorporating a Hill-type law that couples active cytoskeletal mechanics to cell-edge velocity, explaining the counterintuitive experimental observation that traction forces transiently decrease during rapid hypotonic swelling despite an increase in cell size. We further construct a kinematic-geometric phase diagram that connects classical quasi-static theories with our dynamic model. Moreover, we show that traction-force dynamics are sensitive to the loading rate and amplitude of osmotic shock. In addition, stiffness-associated simulations and supporting perturbation evidence suggest a cellular stiffness-associated recovery trend in which stiffer cells exhibit faster volume recovery driven by stronger recoil of hydrostatic pressure.

Ligand residence time is increasingly recognized as an important determinant of drug efficacy. In the human adenosine A2A receptor (hA2AR) bound to the high-affinity antagonist ZMA, mutations at the extracellular entrance of the binding pocket markedly accelerate ZMA dissociation while largely preserving binding affinity. This effect has been attributed to disruption of the E169ECL2-H7.29 salt bridge that constrains the extracellular entrance in the wild-type receptor. However, how the interaction network is reorganized after salt-bridge disruption remains unknown. Here, we combine well-tempered metadynamics and infrequent metadynamics simulations to characterize the ZMA dissociation pathway and transition-state ensemble. The simulations reproduce the experimentally observed trend in dissociation rates and reveal that the hydrogen-bond network stabilizing ZMA in the bound state is largely replaced by water-mediated interactions during unbinding. In the mutants, disruption of the E169ECL2-H7.29 interaction expands the binding pocket and increases hydration, while additional ligand-lipid interactions emerge specifically at the transition state. These interactions are substantially less frequent in the wild type, indicating that membrane lipids can directly participate in ligand escape when the extracellular gate is disrupted. Thus, the mutations selectively reshape the transition-state interaction network including recruitment of membrane lipids. Thus, the interaction of the latter with the ligand accompanies and may contribute to the mutants reduced dissociation barrier, thereby facilitating ligand release.

Diffusion within the cytoplasm is fundamental to numerous biological processes. Fluorescence recovery after photobleaching (FRAP) is one of the most common method for quantifying molecular diffusivity in living cells using standard laser scanning confocal microscopy (LSCM). However, accurately measuring fast cytoplasmic diffusion (typically >10 m^2/s) is challenging due to rapid recovery kinetics, weak signal-to-noise ratios, post-bleach signal artifacts, and spatial restrictions affecting normalization. While individual challenges have been addressed in specific contexts, a simple and robust framework to quantify cytoplasmic diffusivity remains elusive. Here, we present a FRAP methodology specifically designed to overcome these obstacles. By utilizing the Gaussian function -- the impulse response (ImpRes) of the diffusion equation in an infinite medium -- our approach leverages the full spatiotemporal dataset through a single-equation three-parameter fitting procedure, thus releasing restrictions to small regions of interest and arbitrary initial time-points. The methodology was validated on three datasets of increasing complexity: in silico simulated recovery profiles, in vitro data from FITC-dextran in glycerol solution, and live-cell imaging of free cytoplasmic GFP. Systematic comparison with existing models demonstrates that the ImpRes approach significantly reduces sensitivity to noise and imperfect fluorescence normalization, while remaining robust against short-term biases, such as transient probe photo-activation. Given its robustness under realistic experimental conditions and its ease of implementation, the proposed FRAP methodology provides a reliable tool for quantitative cytoplasmic analysis.

P-type ATPases maintain cellular metal homeostasis by coupling ATP hydrolysis to the active translocation of ions across biological membranes. Within this superfamily, P1B -type ATPases play a central role in the transport and detoxification of transition metals such as Cu+ and Ag+. Despite the availability of several crystallographic structures for bacterial ATPases, molecular determinants governing ion selectivity, coordination dynamics, and transport efficiency are not yet fully resolved, particularly for plant homologs, whose conformational landscapes have not been explored in detail at atomistic resolution. In this study, we combine molecular dynamics (MD) simulations with enhanced sampling through metadynamics to characterize the conformational dynamics and ion-binding mechanisms of representative heavy-metal P-type ATPases. By comparing bacterial and plant homologs in phosphorylated and dephosphorylated states, we examine how phosphorylation is associated with changes in structural plasticity, channel accessibility and ion coordination environments. The simulations indicate phosphorylation-dependent rearrangements of the cytosolic domains and transmembrane helices, which may alter access to the metal-binding pathway. Enhanced sampling identifies distinct conformational basins and possible transition pathways along functionally relevant collective variables, allowing characterization of the free-energy landscape associated with ion displacement within the transmembrane region. Together, these results provide an atomistic perspective on the relationship between metal coordination and protein conformational dynamics. Overall, our findings support a model in which phosphorylation state, structural dynamics, and local coordination environments collectively contribute to the differential behavior of metal ions in P1B -type ATPases.

Centrosome positioning is governed by mechanical interactions between the actin and microtubule cytoskeletons, yet the contribution of different actin architectures and their attachment to the cell membrane remains unclear. We use numerical simulations to study a centrosomal array of microtubules interacting with cortical, bulk, and centrosome-associated actin networks, including passive cross-linkers, motor proteins, and ERM-mediated coupling of actin to the cell membrane. We find that bulk and centrosome-associated actin alone are insufficient for robust centrosome centering, whereas sufficiently dense cortical actin promotes centering through steric interactions with growing microtubules. ERM-mediated attachment of cortical actin to the cell membrane enhances this effect, allowing stable centering at lower actin densities. In a two-centrosome system, microtubule interactions generate a spindle-like structure, while the organization of actin and its membrane attachment through ERM regulate centrosome separation. Antagonistic microtubule motors further limit centrosome separation and establish a finite spindle length. These results demonstrate that ERM-mediated coupling of actin to the cell membrane provides an important mechanical link between the actin cortex and microtubule-driven centrosome positioning, influencing both centrosome centering and spindle geometry.

During the influenza viral life cycle, the viral glycoprotein hemagglutinin (HA) mediates binding, entry, and fusion. Densely packed clusters of HA trimers at the plasma membrane are required to produce infectious virions; however, the mechanism of HA clustering is still unknown. We have shown previously that HA co-clusters with and modulates phosphatidylinositol 4,5-bisphosphate (PIP2) in host cell plasma membranes (PM). Here, we further characterize the relationship between HA and PIP2 using molecular dynamics simulations (MD) and fluorescence photoactivation localization microscopy (FPALM) to elucidate a mechanism of HA-PIP2 interaction. We found that the interaction occurs largely between the PIP2 head group and the cytoplasmic tail domain (CTD) of HA. Mutations of the CTD were made to alter charge (HARE, HARREQ), palmitoylation sites (HAMAY), or a combination thereof (HAREMAY, RREQMAY). MD showed that HARREQ and RREQMAY had the strongest effect on HA-PIP2 interactions through a depletion in the radial distribution function of PIP2 around HA at distances [≤]2.5 nm. FPALM revealed that HA cluster density at the PM was significantly reduced by CTD mutations, with the largest reduction occurring in mutants where the CTD charge and acylation were both altered (HAREMAY). HAREMAY clusters were also found to have larger circularities and perimeters, implying a structural change to the clusters. Mutations in the HA transmembrane domain also caused modest changes to the cluster properties of HA and its co-clustering with PIP2. FPALM showed PIP2 co-clustering with HA was also affected by HA mutations with more free PIP2 localized under HAREMAY clusters. A chemical model of simultaneous HA-PIP2 and PH-PIP2 binding enables interpretation of HA-PIP2 interactions and reveals quantitative differences between PIP2 binding by HA CTD mutants. We conclude that the mechanism of HA-PIP2 interaction consists of at least electrostatic and hydrophobic components. Our insights into the mechanism of HA-PIP2 interaction, and the prevalence of putative PIP2-interacting domains in a number of viral spike proteins suggest it may be fruitful to identify methods of disrupting interactions between phosphoinositides and viral proteins.

In the active loop extrusion model, the cohesin protein complex creates chromatin loops in eukaryotic cells. Extrusion maintains topologically associated domains (TADs), which are contiguous segments of chromatin that preferentially colocalize in space and are typically bounded by CTCF proteins that pause cohesin translocation. Here, we model active loop extrusion with hybrid molecular dynamics - Monte Carlo simulations in entangled flexible linear polymer melts. Intra-chain contact probabilities of polymers with active loop extrusion are enhanced compared to their equilibrium, passive counterparts. Extrusion causes the size of chain segments to be much smaller than in passive melts. While the overlap parameter in passive melts without extrusion monotonically increases with segment length, it is nonmonotonic in active melts and on the order of unity within the parameters of this study. Active loop extrusion suppresses contacts between TADs in favor of intra-TAD contacts. Reduction of overlaps between chain segments dilutes entanglements in active melts. Depending on parameters, active extrusion without TADs may induce more compact conformations than with TADs, due in part to fractal loopy globule-like dynamics. This work suggests that active loop extrusion reduces overlaps between TADs, contributing to effective gene regulation by cis-regulatory elements.

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.

Acetylcholine receptors (AChRs) expressed at the nerve-muscle synapses are prototypic allosteric receptors that shuttle between a resting Closed (C) and active Open (O) states. ACh binding at the neurotransmitter binding sites (TBS) leads to opening of the gate in the channel pore that regulates ion flow. How agonist binding to the TBS communicates the allosteric signal to the channel gate located [~]50 [A] away is not understood. In the absence of agonists, the wild-type and mutant AChRs, including those causing congenital myasthenia syndrome, show constitutive gating. However, whether allosteric activation pathways in the presence vs absence of agonists are identical, is debatable. Here, by using a combination of kinetic modelling, phi ({phi})- and activation energy ({Delta}{Delta}G[palclick]) estimation for >60 residues from single channel current recordings and molecular dynamics simulations, we show the existence of parallel gating pathways ( major and minor) in the unliganded AChRs and activation pathways are non-identical for the liganded vs unliganded AChRs. Kinetic analysis of the minor gating and correlation between the state residence probabilities vs C-loop conformations suggest, 1. the C-loop capping triggers allosteric communication independent of the presence of an agonist and, 2. the minor gating is a pre-existing allosteric pathway which is preferentially chosen in the presence of agonists. Further, we show the presence of a continuous live-wire like allosteric network in the liganded receptor between the TBS and the gate constituted by residues which lower the activation energy barrier by >-4 kcal/mol. In contrast, in the unliganded AChRs, the major gating seems to initiate from hub residues in the allosteric network. The results presented here provide novel insight into the mechanism of allosteric signal initiation and communication in AChRs.

Polyvalent-cation-induced condensation and reentrant transition in double-stranded DNA are classically described by backbone electrostatics. Whether the same principles govern single-stranded DNA, whose solvent-exposed bases can interact within and between chains to form complex phases, is unknown. Here, we employ simulations and experiments to show that the tetravalent cation spermine (Spm4+) drives two equilibrium transitions in poly(dT) and poly(dA). The onset of phase separation, resulting in thermodynamic equilibrium between the dispersed low-density phase and high density coacervate, occurs at a low sequence independent Spm4+ concentration and is primarily driven by charge balance between phosphate groups and Spm4+. Strikingly, the condensed phases are stabilized by at most 1.5 (poly dT) and 2.5 (poly dA) bridging Spm4+ per chain. The ssDNA chains condense to form fluid-like networks without chain collapse. The fluidity of chains in the dense phases is confirmed by fluorescence recovery after photobleaching experiments. Both simulations and experiments show that the range of spermine concentration over which phase coexistence persists is sequence dependent and nearly threefold wider in poly(dA). At higher sequence dependent spermine concentrations, a reentrant phase, characterized by dissolution of the coacervates, emerges. The transition to the reentrant phase occurs when the number of bridging Spm4+ molecules vanishes at high sequence dependent spermine concentration. The combination of experiments and simulations show that the two phase transitions in ssDNA are driven by an interplay of stacking and electrostatic interactions.

Clathrin-mediated endocytosis (CME) is the primary pathway for internalization of extracellular and membrane cargo in eukaryotic cells. It is characterized by a patch of the plasma membrane invaginating and pinching off to generate a cytoplasmic vesicle. Recently, experimental evidence has begun to accumulate in support of proteins assembling into liquid-like condensates at nascent CME sites, but potential functional consequences are not understood. Given that condensate capillary forces can influence membrane bending, we constructed complementary analytical and numerical model systems to probe the mechanical influence of a protein droplet on endocytosis. Model evaluations and analysis reveal that a droplet can regulate the progression of endocytosis by stalling invagination until the droplet grows past a threshold size. After the droplet-mediated brake is released, the droplet provides a force that can drive the membrane bud toward scission. Thus, we propose a ``mechanical timer'' mechanism as a function for protein phase separation in cellular membrane trafficking.

We used simulations of short single stranded DNA (ssDNA) homopolymers, based on the sequence dependent Three Interaction Site (TIS) model, to calculate the persistence length (lp) in polyvalent cations. The TIS model accounts for stacking interactions and electrostatic interactions are treated using the Coulomb potential. We find that lp for dT30 (T is thymine) and dA30 (A is adenine) is quantitatively fit using [Formula] ([Formula] is the bare persistence length,{lambda} is a dimensionless constant, and{kappa} -1 is the inverse Debye length) in the divalent cations Mg2+ and Ca2+. The dependence of lp on{kappa} is surprising because it was derived for long flexible polyelectrolytes in which the charges interact via the Debye-Huckel potential. The [Formula] values are 0.4 nm and 1.1 nm for polyT and polyA, respectively. Strikingly, lp is almost independent of the tetravalent spermine concentration. There is no clear theoretical explanation although simulations suggest that the number of spermine molecules that bind to the ssDNA saturates at a small value. A qualitative picture, based on the restrictions of access to the phosphate groups due to volume exclusion of the anisotropic structure of Spm4+, rationalizes the simulation results. The predicted dependence of lp in spermine awaits experimental test.

It is important to determine whether native state dynamics drive the misfolding and oligomerization of the prion protein, which are important events in prion disease, and how they are modulated by conformational conversion. Native (N) mouse prion protein (moPrP) is known to form small (OS) and large (OL) oligomers rich in {beta}-sheet, and in this study, photoinduced electron transfer-fluorescence correlation spectroscopy (PET-FCS) has been used to characterize intramolecular dynamics within individual monomeric units in both isolated OS and OL, as well as the diffusion properties of the oligomers. It is estimated that OS and OL comprise of about 15 and 55 monomeric units, respectively. Microsecond dynamics at each of the two regions that are the 1-3 and 2-3 interfaces of native protein are distinct in N, OS and OL, although they occur on very similar timescales. Analysis of the evolution of the distribution of diffusion times, determined using the maximum entropy method, indicates heterogeneity in the oligomerization reaction. Analysis of the change in the fluctuations which occur in two different timescales in the native state ensemble shows that they are damped more at the erstwhile 1-3 interface than the erstwhile 2-3 interface. The difference in the extent of damping at the erstwhile 1-3 and 2-3 interfaces can be explained on the basis of the structural changes known to occur across each region. The changes in dynamics occur concurrently in both regions, indicating that the structural changes accompanying conformational conversion also occur simultaneously during the oligomerization of moPrP.

Inefficient endosomal escape is a crucial barrier to intracellular delivery of nucleic acid therapeutics using lipid nanoparticles (LNPs). The use of ionizable lipids (ILs) has significantly improved cargo delivery efficiency, yet the physical basis of their interaction with endosomal membranes and their role in endosomal escape remain unclear. It has been suggested that, as ILs become cationic during endosomal acidification, electrostatic affinity promotes fusion of the LNPs with the endosome. In this paper, we propose an additional mechanism in which ILs are redistributed from LNPs to host membranes, modulating the elastic properties and curvature of the membrane, lowering the energetic threshold for endosome disruption. To test this, we quantified the spontaneous curvature of clinically relevant ILs and ATP-binding lipids and measured the membrane mechanics of giant unilamellar vesicles (GUVs) with an endosome-relevant composition at endosome-relevant pH. Small-angle X-ray scattering (SAXS) measurements reveal that the incorporation of ILs and ATP lipids into endosome-mimetic membranes shifts the spontaneous curvature towards more negative values. Micropipette aspiration experiments indicated a decrease in the apparent area compressibility modulus of membranes doped with ILs and ATP lipids. In addition, membranes showed enhanced fluctuation amplitudes and altered relaxation behavior, consistent with membrane perturbations associated with lipid insertion and pH- or ATP-driven destabilization. Under conditions promoting the partitioning of ILs or ATP-binding lipids, we further observed reduced bending rigidity and increased heterogeneity in membrane tension. Together, these results support a model in which ILs (as well as newly developed ATP-binding lipids) partition into endosomal membranes, softening the membrane and generating local curvature frustration that facilitates endosomal disruption during the natural acidification process. By quantitatively linking lipid composition with changes in membrane elasticity and fluctuation dynamics, this work provides a biophysical framework for understanding how lipid redistribution may contribute to endosomal escape and improve delivery efficiency.

Many membrane proteins contain intrinsically disordered regions (IDRs) that play key biological roles by providing structural plasticity, harboring sites for post-translational modifications, and mediating protein clustering and phase separation. Residue-level molecular models parameterized against experimental data have provided insights into how IDR sequence controls conformational properties and phase behavior in soluble proteins. Here, we extend this modeling framework to membrane-associated IDRs. We adapt a coarse-grained lipid model, iSoLFv2, for four phospholipids and combine it with CALVADOS, a residue-level model for IDRs and multi-domain proteins. Protein-lipid cross-interaction parameters are calibrated to reproduce predicted insertion and orientation of transmembrane proteins with diverse architectures. We validate the resulting model against Wimley-White free energies of transfer of hydrophobic peptides and against an experimentally refined conformational ensemble of a flexible membrane receptor. Finally, we show the applicability of the model to a membrane-associated assembly of signaling proteins. The model provides a computationally efficient framework for studying conformational ensembles and assembly of proteins at bilayer-water interfaces.

Recent experiments show that protein condensates sitting on opposite surfaces of a flat lipid membrane move together and prefer to overlap, even though they cannot touch each other. This points to an indirect, membrane-mediated interaction. Two mechanisms could be responsible: a curvature-induced interaction, which is energetic in origin, and a fluctuation-induced interaction, which is entropic. Here we study both with coarse-grained molecular dynamics simulations, using Cookes implicit-solvent lipid model together with a generic bead-spring polymer model for the condensate. We compute the potential of mean force between two condensates across the membrane. For condensates of the same size, full overlap is unfavorable, and the pair instead settles into a partially overlapping state that bends the membrane into an S-like shape. When the two condensates differ strongly in size, full overlap becomes favorable. We explain this with a simple geometric picture. The condensate wets the membrane as a thin film and imposes curvature only along its rim, while membrane tension flattens the membrane under its interior. The resulting ring of curvature can trap a smaller condensate on the opposite side. We also compare the bending undulations and the effective bending modulus of a bare membrane, a membrane with one condensate, and a membrane with condensates on both sides. A wetting condensate suppresses the undulation modes and stiffens the membrane, but whether this makes overlap entropically favorable remains inconclusive. Our results indicate that the coupling is driven mainly by curvature, and that it depends on the wetting mechanism and on the membrane tension.