The eukaryotic genomes encode hundreds of proteins that function as ion channels and transporters. Essential for sustaining life, these proteins mediate the movement of inorganic ions (e.g., K+, Na+, Cl-, and Ca2+) across the plasma membrane according to their electrochemical gradients. In multicellular organisms, a diverse array of ion channels contributes to the maintenance of the resting membrane potential, the regulation of pH, osmolarity, and cell volume, and the control of secretion, electrical excitability, and synaptic activity, among many other fundamental physiological processes. Although independent evolutionary origins have been proposed for several ion channel families, their relative hierarchical importance for cellular viability remains poorly understood. To advance our knowledge of ion channel evolutionary history, we focused on determining the minimal combination of permeabilities that allows cellular viability. To this end, we conducted a survey of representative prokaryotes with small genomes across bacterial and archaeal phyla. By focusing on the smallest genomes, our approach enabled the identification of five ion channel architectures shared among prokaryotes. Among these, non-selective mechanosensitive channels (MscS and MscL) are the most abundant, followed by potassium channels, CLC-type channels and proton channels of the MotA/TolQ/ExbB family. The conservation of the mechanosensitive protein architecture across archaeal and bacterial membranes suggests that the capacity to monitor physical membrane integrity predates the requirements for electrical communication.

Biological membranes contain a diverse set of membrane proteins surrounded by many different lipids, and the lateral organization and function of these molecules are closely intertwined. Here, we use coarse-grained molecular dynamics (MD) simulations to explore how hydrophobic mismatch between the length of transmembrane (TM) proteins and the thickness of the surrounding lipid membrane impacts the spatial distribution of the lipids. We constructed idealized cylindrically symmetric proteins, inspired by the "Mattress Model" developed in the 1980's, and simulated these model proteins in different lipid compositions. We found that unsaturated lipids were attracted to short TM proteins that thinned the membrane, while fully saturated lipids were attracted to long TM proteins that induced membrane extension. A simple mechanical description of the membrane deformation energy coupled to a lipid mixing model accurately predicted the enrichment/depletion, which was up to 33% in some cases. Overall, we find that the main driver behind unsaturation-based sorting is the effective lipid length that conforms to match the hydrophobic thickness of the TM protein. Additionally, our simulations highlight that lipid sorting behavior is sensitive to protein tilt and protein surface roughness. By teasing out the fundamental physical principles in these simple models, our results provide a foundational understanding of how proteins and lipids form complex and transient assemblies, which we believe will be important for interpreting lipid-protein interactions for a host of membrane proteins that regulate cellular membranes and cell function.

Ca2+ binding regulates muscle contraction through coupled structural and dynamical mechanisms, yet a unified description of mutation-induced perturbations in binding energetics remains incomplete. Mutational effects on proteins are often high-dimensional and difficult to interpret mechanistically. Here, we develop a physically interpretable low-dimensional reaction coordinate that captures coordination environment, electrostatic features, and dynamical coupling. Structural, electrostatic, and dynamical descriptors correlate with experimental binding free energies with Pearson coefficients of Rp = 0.83, 0.83, and 0.85, respectively. Integration of these domains improves agreement with experiment (Rp = 0.90), indicating substantial dimensional compression of the underlying descriptor space. This framework provides a physically grounded description of how mutations reshape Ca2+ binding energetics in a minimal EF-hand system.

The SARS-CoV-2 main protease (MPro) is an essential enzyme for viral replication and a primary target for antiviral drug development. Despite extensive structural and biochemical characterization, the allosteric mechanisms by which dimerization informs conformational changes at active site lack an explicit comparison across the different states that identify key residues that connect substrate binding, dimerization, and catalytic activation. Here, we integrate microsecond time scale all-atom molecular dynamics (MD) simulations with dynamical network analysis to characterize how ligand binding and dimerization modulate the allosteric communication landscape of MPro. We performed triplicate 1-μs simulations of MPro in the monomer and dimer states. For each of these states, we simulated MPro in the apo state, as well as bound to a natural peptide substrate (nsp 15/16), the covalent inhibitor nirmatrelvir (Paxlovid) and the noncovalent inhibitor ensitrelvir (Xocova). Dynamical network analyses from the resulting simulations reveal that dimerization redirects the highest correlated motions from the interdomain loop towards the domain II and III interface. At the dimer interface, we identified N-terminal and domain II β-hairpin residues that act as central communication hubs creating networks that connect both chains in the dimer. Small molecule binding to the active site further modulates these networks in distinct ways: nirmatrelvir and peptide substrate binding results in the formation of allosteric networks within the oxyanion loop, while ensitrelvir-bound monomeric MPro results in a dimer-like network, suggesting an inhibitory "allosteric switch" mechanism that may hinder dimerization upon binding. Across all systems, domain III emerges as an allosteric "pivot", providing a platform that allows the most relevant networks to connect inter-chain communication to the active site upon dimerization. Together, these findings define how correlated motion networks couple active-site dynamics to dimerization and ligand binding, providing molecular insight into the principles governing allosteric regulation in MPro. This framework highlights potential avenues for developing antivirals that target not only the catalytic site but also the communication pathways sustaining dimer stability and enzymatic function.

Most cancer deaths result from metastasis, yet only a subset of tumor cells can complete this process. Polyaneuploid cancer cells (PACCs), which arise via endoreplication under stressors such as hypoxia, are implicated as metastatic drivers, but how they acquire this capacity remains unclear. Here, we show that under extreme self-generated hypoxia, the polyaneuploid subset of PC3 prostate cancer cells upregulates a hypoxia-responsive, HIF-1α-dependent RhoA signaling axis that promotes aerotaxis, a behavior predictive of intravasation during metastasis. We observe an enrichment of PACCs using a membrane-based culture system in which extreme, self-generated radial oxygen gradients emerge as cellular oxygen demand exceeds supply, recapitulating key features of the tumor microenvironment. Under these conditions, PACCs exhibited elevated HIF-1α and RhoA expression relative to non-PACCs. Disruption of either HIF-1α or RhoA attenuates aerotaxis, with RhoA expression suppressed upon HIF-1α inhibition, suggesting a functional HIF-1α- dependent RhoA pathway selectively enhanced in the PACC state. Quantitative analysis of nuclear morphology further reveals that nuclear circularity and solidity, morphology metrics associated with nuclear rounding and boundary organization, increase with and saturate at high nuclear area. Notably, the hypoxic PACC population exhibits significantly greater nuclear circularity and solidity compared to non-PACCs, suggesting increased rounding and stabilization of the enlarged nuclear architecture characteristic of PACCs under extreme hypoxia. Together, these findings reveal a new functional role for HIF-1α- dependent RhoA signaling in regulating aerotaxis in polyaneuploid cancer cells, which may enable aerotactic escape from hypoxic tumor cores and entry into oxygen-rich vasculature. Additionally, the association between elevated RhoA signaling and increased nuclear circularity at larger nuclear area suggests a potential link between hypoxia-responsive migration and maintenance of enlarged nuclear architecture in the PACC state. Collectively, these characteristics highlight PACCs as a hypoxia-adapted subpopulation with relevance for anti-metastatic therapeutic strategies.

Molecular dipoles near interfaces emit highly directional radiation due to near-field inter-actions, making surface-bound fluorophores sensitive probes of local physicochemical changes. We intro-duce "smart coverslips", standard microscope substrates, which are stably coated with uniform, brightly fluorescent nano-bead films, that exploit refractive-index-dependent shifts in the radiation pattern for sensitive micro-refractometry in small volumes. Supercritical-angle fluorescence refractometry uses single back-focal-plane images to allow us real-time RI sensing and nanometric thin-film height measurements without the need for multi-angle or multi-wavelength acquisition. Our fast, label-free, and non-invasive approach allows measurements of thin-film properties and monitoring of interfacial dynamics on a standard inverted microscope and is broadly applicable to nano-biophotonics, chemical sensing, and in-situ materials analysis. Applied to membrane biophysics, these findings establish a versatile approach for resolving the nanoscale organization of membrane-proximal or -associated structures.

Photosynthetic microorganisms rely on multiple central-carbon-metabolism pathways to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes that operate across disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. Here, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial, subcellular organization of key carbon fixation enzymes and genetic information processes coupled to a non-spatial genome-scale metabolic model (GSMM). We combine perturbative, time-resolved multi-omics measurements and cryo-ET derived 3D segmented volumes as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulate carbon fixation by controlling the structural assembly of a protein megacomplex, the "dark complex," at a conserved regulatory node of the Calvin-Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates the importance of spatial heterogeneity in understanding phenotypic robustness. Spatiotemporal sequestration creates a timing hierarchy spanning seconds to hours and noise-buffering behavior that cannot be recovered from well-mixed phenomenological models or purely time-resolved descriptions. Constrained by spatial heterogeneity, local enzyme stoichiometry and diffusion-limited assembly/disassembly determine effective stochastically varying control kinetics. Diffusion-driven fluctuations amplify transcription of highly expressed genes, whereas PTM-dependent regulation on enzyme stoichiometry maintains perturbation-driven phenotypic outcomes. 4D whole cell modeling with perturbation-based multi-modal experiments unlocks the ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and light-dependent central carbon metabolism. The outcome of this work addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4.

Multidomain proteins connected by flexible linkers populate conformational ensembles that are challenging to characterize using conventional structural biology methods. In domain-linker-domain (DLD) proteins, linker-mediated inter-domain relative positions and orientations are functionally relevant, yet their dynamical behavior in solution normally remain poorly described. Small-angle X-ray scattering (SAXS) provides ensemble-averaged structural information for such systems; however, coupling with computational modeling is required to accurately describe the dynamic behavior of this family of proteins in solution. Here, we present a systematic evaluation of five ensemble-generation strategies applied to a set of eighteen proteins sharing the same two globular domains, connected by naturally occurring linkers of varying length and composition. Modeling methods based on different underlying principles are compared by assessing their agreement to experimental SAXS data, showing a large disparity and systematic structural biases among them. Furthermore, for each approach, we examine the effect of refinement against SAXS restraints and assess its capacity to describe the experimental data, as well as the induced biases in global dimensions and inter-domain distance distributions. This analysis underlines the importance of the initial conformational pool for deriving experimentally compatible ensembles. Overall, this work provides a high-quality benchmark for SAXS-driven ensemble modeling of flexible, multidomain proteins and establishes a framework for the critical interpretation of solution scattering data in systems with pronounced conformational heterogeneity.

Although traditional molecular dynamics simulations successfully capture a variety of different molecular interactions, the protonation states of titratable residues are kept static. A recent constant-pH molecular dynamics implementation in the GROMACS package allows pH effects to be captured dynamically, and promises to provide both the accuracy and computational performance required for studying pH-mediated conformational dynamics in large, complex systems containing hundreds of titratable residues. Here, we demonstrate the applicability of this constant-pH implementation by simulating the proton-gated ion channel GLIC at resting and activating pH, starting from closed and open structures. Our simulations identify residues E26 and E35 as especially pH-sensitive and reveal state-dependent pKa shifts at multiple residues, as well as side chain and domain rearrangements in line with the early stages of gating. Our results are consistent with several previous experimental findings, demonstrating the applicability of constant-pH simulations to elucidate pH-mediated activation mechanisms in multidomain membrane proteins, likely extensible to other complex systems.

Integrins are bidirectional mechanochemical receptors that transmit signals upon ligand binding to the cytoskeleton (outside-in) and cytoskeletal forces back across the membrane (inside-out) to the integrin-ligand bond. Integrins are activated prior to ligand binding, which involves large conformational rearrangements across the extracellular, transmembrane, and cytoplasmic regions. While the conformational and energetic basis of outside-in activation is increasingly well defined, the mechanical forces required for separating the tightly packed αβ transmembrane (TM) helices during inside-out signaling remain largely unknown. Here, we directly quantify the forces required to dissociate integrin TM domains (TMDs) in a lipid environment. Engineered α5β1 polypeptides consisting of TMDs and cytoplasmic tails were reconstituted into lipid nanodiscs and probed using single-molecule optical tweezers. Mechanical marker domains on each cytoplasmic tail verified correct vectorial force application, and fluorescent lipids confirmed nanodisc integrity. We find that the heterodimeric TM complex is a mechanically robust unit. In wild-type constructs, no TMD separation was observed in repeated pulls up to ∼35 pN. Point mutations in the β1-TMD (G744L, L748R) that weaken TMD interactions revealed discrete splitting events. The high mechanical forces necessary for TMD separation support a "ratchet-like" role for mechanical forces in inside-out signaling: rather than actively opening closed TMDs they prevent re-closing of spontaneously split TMDs thus keeping them open and activated.

The homotrimeric SARS-CoV-2 spike glycoprotein comprises two subunits: S1, which recognizes host-receptors through its receptor-binding domains (RBDs), and S2, anchored to the viral membrane through its transmembrane domain (TMD), which facilitates the fusion of the viral envelope with the host cell membrane. Upon host-receptor engagement and proteolytic activation, S1 dissociates and triggers a large conformational transition in S2, involving structural rearrangements in the S2 ectomembrane-domains and the TMD. While studies have focused on the ectomembrane-domain dynamics, the TMD has typically been modeled as being in a trimeric state. Here, we use molecular dynamics simulations of a coarse-grained structure-based model (SBM) with an implicit membrane to investigate the role of TMD dynamics in modulating S2 conformational conversion. We first recapitulate previous results from an all-atom SBM with a trimeric TMD and re-emphasize that the extended pre-hairpin intermediate state of S2, which brings the two membranes into apposition, is a byproduct of the prefusion-to-postfusion transition. Next, by introducing dynamics into the TMD, we find a late fusion intermediate structurally similar to a recent cryo-EM structure. A dynamic TMD also makes the conformational transition faster. Simulations including the S1/S2 complex reveal coupled RBD-TMD dynamics: when all three RBDs are in the closed state, they can stabilize the TMD in a trimeric configuration, whereas the opening of a single RBD can trigger a transition to a dynamic TMD. So, the dynamics and the conformational preferences of the TMD can be tuned by the presence and conformation of S1. There is some evidence that the TMDs of class I viral fusion proteins, such as spike, contribute to viral fusion by modulating membrane properties. Our simulations indicate an expanded role for the function of the TMD, where it can directly regulate the kinetics of S2 conformational transitions.

Several members of the adhesion subfamily of G protein-coupled receptors (aGPCRs) are capable of self-activation by an internal agonist sequence (i.e., the Stachel) that is exposed upon removal or conformational changes of the N-terminal fragment of the receptor. Synthetic peptides derived from the Stachel sequence can be used as exogenous agonists. In the inactive form of the full-length receptor, the Stachel is sequestered as the β13 strand within the G protein-coupled receptor (GPCR) autoproteolysis-inducing (GAIN) domain, but it engages the seven transmembrane region as a helix when it is either an intramolecular sequence or a synthetic peptide. Little is known about the molecular details underlying this transition, but we hypothesize that a disordered conformation is central to this intermediate state in receptor activation. Despite the primarily helical Stachel AlphaFold3 and PEP-FOLD4 models predicted with high confidence for the entire aGPCR subfamily, computational predictions and biophysical experiments reveal a predominantly disordered conformation in solution. Investigating the ADGRG6 (also known as GPR126) Stachel peptide, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments reveal a predominantly random-coil conformation in aqueous buffer, polar detergent micelles, and zwitterionic lipids. Titration of trifluoroethanol uncovered a two-state equilibrium between an unfolded and helix-containing conformation with NMR localizing a single-turn helix to residues L846-L849. Taken together, these data indicate the ADGRG6 Stachel peptide is primarily disordered with a subset adopting partial helical structures, likely requiring the steric hindrance of the receptor binding pocket to fully induce helix formation in an induced-fit mechanism.

Cancer cells in hypoxic environments often proliferate less but exhibit enhanced migration relative to their normoxic counterparts. Recent in vitro and in silico studies have characterized the role of hypoxic memory - the ability of cancer cells to retain their hypoxic phenotype even when reoxygenated - in tumor invasion. However, the observations have been limited either to exposing cancer cells to hypoxia for a fixed duration or by assuming a fixed-time persistence of the hypoxic state upon reoxygenation independent of the duration of hypoxia exposure. Thus, duration-dependent cell-state changes during hypoxia and their impact on hypoxic memory remains unclear. Here, we first analyze transcriptomic data from breast cancer samples to show that the genes upregulated at transcriptional level and hypomethylated at epigenetic level are enriched in cell invasion, indicating hypoxic memory-driven process of tumor invasion. Next, we used a computational model to investigate how the spatial-temporal dynamics of oxygen levels in a tumor drive duration-dependent changes in hypoxic memory and influence tumor invasion dynamics. Our simulation results show that such dynamic hypoxic memory can drive enhanced tumor invasion over a fixed hypoxic memory by a) enriching hypoxic cell density at the tumor front, b) reducing sensitivity of hypoxic cell state to fluctuations in oxygen supply, and c) enhancing effective diffusion of hypoxic cells. Our results highlight the crucial role of dynamic hypoxic memory in shaping tumor invasion dynamics, underscoring the need to elucidate its underlying mechanisms in future studies.