- by Lin, Y.-C., Luo, S., Patil, A., Primanis-Erickson, N., Jiang, X., Webb, B., Hsu, K.-L.Phosphofructokinase-1 (PFK-1) catalyzes the ATP-dependent conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6BP), the first committed step of glycolysis. Beyond classical allostery, the liver isoform PFKL forms higher-order assemblies, but how ligand binding redirects intermolecular interactions remains unclear. Here we use high-speed atomic force microscopy (HS-AFM), topology-based AFM image simulations, and molecular dynamics (MD) simulations to define ligand-dependent assembly switching of human PFKL. Wild-type PFKL (PFKL WT) forms lattice-like assemblies under APO and ATP conditions, whereas coordinated ATP and F6P loading […]
- by McArthur, S. J., Umeda, K., Kodera, N.Although the maturation state of dengue virus (DENV) particles is a key determinant of their infectivity, maturation is unusually inefficient. Fully mature and immature DENV particles are well-studied; however, little is known about partially mature particles. Moreover, single-particle structural dynamics and nanomechanical properties are unknown. Here, we observe wildtype and immature DENV particles using a single-particle approach combining high-speed AFM (HS-AFM) and 3D force mapping (3DFM). HS-AFM shows that the conformations of each morphotype are heterogeneous and dynamic in liquid, […]
- by Ngo, T., Faiyazuddin, M., Nguyen, T. D., Haug, J., Shen, Q., Gałecki, S., Borges, H. M., Chen, B., Wang, X., Zhu, H., Pappas, S. S., Voigt, F. F., FIolka, R., Dean, K. M.Altair-dvOPM is an open-access direct-view oblique plane microscope designed for large-field, three-dimensional imaging of cleared and expanded tissue sections. By combining photographic-lens-based detection, externally launched oblique illumination and precision-registered modular baseplates, the system achieves micrometer-scale lateral resolution over a ~5.4 mm field of view without custom objectives or highly specialized alignment procedures. We demonstrate imaging across scales, from subcellular structures in expanded cells to centimeter-scale expanded tissue sections, and provide documentation, CAD files, Zemax models and open-source control software to […]
- by Uchiyama, T., Ueno, H., Sobti, M., Furlong, E. J., Brown, S. H. J., Stewart, A. G., Noji, H.F1-ATPase achieves unidirectional rotation of its {gamma} shaft through coordinated conformational cycling of the 3{beta}3 ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF1 lacking the lower half of the rotor shaft by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six pauses per turn, indicating the presence of an […]
- by Sasai, M., Fujishiro, S.When three cyanobacterial proteins–KaiA, KaiB, and KaiC–are incubated with ATP in vitro, the phosphorylation level of KaiC exhibits stable circadian oscillations. Biochemical and structural analyses have shown that KaiC's ATPase activity is crucial for these oscillations, leading to the hypothesis that ATP-consuming dynamics function as a molecular clock, determining the oscillation period of individual molecules. Moreover, these molecular clocks synchronize with one another, resulting in collective oscillations at the ensemble level. In this study, we develop a theoretical model to […]
- by Mandal, M., Shpinov, Y., Lahlou, A., Pham, F., El Hajji, L., Coghill, I., Laureau, E., Plamont, M.-A., Perez, F., Le Saux, T., Aujard, I., Gautier, A., JULLIEN, L.Reversibly photoswitchable fluorophores are widely used in advanced bioimaging but their design remains demanding. Here, we introduce a new series spanning the whole visible range, which results from combining a large set of fluorogens with the FAST protein scaffold. We first demonstrate that these well-established labeling fluorescent protein tags turn into negative reversible photoswitchers upon decreasing the fluorogen concentration and increasing light intensity. We then show that using not anymore one but two fluorogens adds new responses to illumination. Thus, […]
- by Gyllingberg, L., Haque, A., Ray, S. K., Weber, G., Graham, J. M., Garnier, S.How can simple organisms lacking nervous systems encode and transmit environmental signals to generate complex, adaptive behaviours? Using the unicellular organism Physarum polycephalum as a model, we identify a unifying mechanochemical mechanism that links intracellular calcium oscillations to large-scale behavioural coordination. We first demonstrate experimentally that local perturbation of the actomyosin cortex is sufficient to induce symmetry breaking and directed migration, even in the absence of nutrient cues. Building on evidence linking calcium concentration to actin depolymerization and contractile relaxation, […]
- by Wijerathne, T. D., Chandrasekharan, A., Bhatt, A., Luo, Y. L., Lacroix, J. J.PIEZO proteins (PIEZO1 and PIEZO2) are essential mechanosensitive channels. PIEZO1 is thought to be selectively activated by Yoda molecules (Yoda1 and Yoda2). Although a structural framework for PIEZO1 activation by Yoda1 exists, a molecular mechanism underlying this selective activation is lacking. Here, using electrophysiology and calcium imaging, we show that Yoda1 increases PIEZO2 open probability and stretch sensitivity as efficaciously as PIEZO1 but elicits weaker PIEZO2-dependent calcium entry, rationalizing why its effect on PIEZO2 has been overlooked. Both Yoda1 and […]
- by Umney, O., Curd, A. P., Martin, H., Lewis, T., Tang, A. A.-S., Balusubramanian, H., Khuon, S., Aaron, J., Peckham, M.Sarcomeres, the basic repeating unit of striated muscle, are joined together by crosslinked actin filaments found at the boundaries of muscle sarcomeres, termed Z-discs. Z-discs play a key role in cardiac signalling and disease, however, the arrangement and function of many of the proteins present in the Z-disc remain to be understood. Here, we determined the organisation of 3 key proteins, ZASP, -Actinin-2 and the Z1Z2 epitope of titin, located within the Z-disc. We fluorescently labelled these proteins in cardiac […]
- by Dhillon, H., Lewis, R. S.Following ER Ca2+ depletion, Ca2+ release-activated Ca2+ (CRAC) channels are activated by STIM1 at ER-plasma membrane junctions. The restricted localization and low conductance of the CRAC channel (
- by Majee, A., Merlitz, H., Schiessel, H., Sommer, J.-U.The hierarchical organization of multiphase biomolecular condensates into core-shell architectures is a fundamental problem in soft matter and biophysics. While classical explanations rely on hierarchies of interfacial tension ({gamma}) between coexisting liquids, the ultralow tensions of condensates (0.1-1 N/m) render such hierarchies potentially fragile. We introduce a robust assembly principle based on Polymer-Assisted Condensation (PAC), in which a single polymer species dictates the entire structure. The polymer nucleates a dense core by recruiting a condensation-incompetent protein (P1). A second incompetent […]
- by Savinov, A., Sadasivan, J., White, K. J., Rubien, J. D., Li, G.-W., Case, L. B.Biomolecular condensates are a major driver of cellular organization; however, we lack a predictable and systematic approach to modulate the multivalent interactions underlying their formation. Here, we demonstrate that the AI-driven FragFold method enables robust and generalizable design of protein fragments to control biomolecular condensate formation. We apply this approach across diverse proteins: G3BP1, SARS-CoV-2 nucleocapsid, TDP-43, and focal adhesion kinase (FAK). Computationally screening 2,235 fragments, we selected 18 candidates for further investigation. Overall, we attain a 50% success rate […]
- by Chen, G.-Y., Wu, Z.-Y., Chen, S.-H., Yang, P.Take-off is a fast and energy-efficient strategy for bipedal animals, such as birds, to achieve rapid movement; however, how muscle physiology scales to govern this universal behavior remains unresolved. Research in the physiology of other species is not readily applicable. As a result, important questions, whether theropod dinosaurs such as Tyrannosaurus rex were capable of jumping, remain unanswered. In this article, we coupled Lagrangian dynamics with Hill muscle equations and developed new experimental methods to quantify joint rotational stiffness and […]
- by Lazzari-Dean, J. R., Millett-Sikking, A., Rao, P., Jensvold, Z. D., Baddock, H., Ingaramo, M., Nile, A. H., York, A. G., Preciado Lopez, M.Protein-protein interactions (PPIs) mediate diverse cellular processes, but PPIs are typically characterized using reconstituted in vitro biochemical and biophysical approaches. Current approaches for PPI detection in living cells are limited in the scope of interactions they can capture and often require prior knowledge of the interacting partners. To close this gap, we developed triplet tumbling microscopy (TTM), which reveals the interactions of a tagged protein of interest in cells in real time. TTM reports protein complex size from rotational diffusion […]
- by Gadhe, L., Konstantoulea, K., Mazumder, A., Chen, J., Joachimiak, L. A., Louros, N. N.Amyloid fibrils are intrinsically polymorphic protein assemblies that form distinct structural strains linked to diverse biological and pathological outcomes. Yet, the principles governing how sequence encodes diverse fibril architectures, and the extent to which a given fold constrains underlying amino-acid sequence compatibility, remain poorly understood. Here, we apply generative protein design to directly interrogate the sequence-structure relationship of defined fibril architectures, using -synuclein (S), a protein known to form highly polymorphic amyloid fibrils, as a model system. Sampling sequence space […]
- by Cioffi, M., Luque, A.Adeno-associated virus (AAV) is the preferred viral vector platform in gene therapy. Yet its packaging capacity, about 4.7 kb (kilobases), limits its therapeutic potential and represents a major bottleneck in the field. The packaging capacity of AAV is constrained by its small capsid, which forms a 26-nm-diameter shell assembled from 60 capsid proteins in a T=1 icosahedral architecture. Here, we propose increasing the cargo capacity of AAV vectors by engineering the next possible icosahedral architecture, T=3 (180 capsid proteins), which […]
- by Steinmetzger, C., Karlsson, H., Fontana, C., Galindo Riera, N., Riad, M., Kosek, D., Schlagnitweit, J., Steiner, E., Friebe Sandoz, S., Andersson, E. R., Petzold, K.A-minor motifs consist of adenosines docking into adjacent RNA minor grooves, are the most prevalent 3D interaction stabilizing RNA structures, and widely considered as being static. NMR spectroscopy reveals a secondary structure equilibrium of these motifs between engaged and disengaged states, which we term the A-minor switch. A switch in E.coli ribosome helix 44 consists of a sparsely populated, transient single-nucleotide register shift that sequesters adenosines from their 3D structural A-minor contacts. Mutational trapping of the NMR-defined, A-minor-engaged ground and-disengaged […]
- by Nune, M., Petchiappan, A., Botos, I., Majdalani, N., Shapiro, S. H., Ghirlando, R., Tai, C.-H., Abeykoon, A., Stanley, A. M., Beach, B. M., Gottesman, S., Buchanan, S. K.The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction […]
- by Conde-Torres, D., Garcia-Fandino, R., Pineiro, A.Designing peptide sequences that remain stable and selective across heterogeneous environments remains a central challenge in biomolecular modeling. Here we introduce an interpretable, physics-based Hamiltonian for environment-conditioned design of -helical peptide sequences. The model integrates helix propensities, pairwise interactions, electrostatics, anisotropic solvent exposure, and interfacial geometry into a unified energy function. To enable comparison across sequence lengths and environments, all contributions are rescaled and expressed as Z-scores relative to random sequence ensembles, yielding a normalized design landscape with balanced physical […]
- by Shang, X., Bartels, N., van der Voort, N., Neusch, A., Salama, N., Thaventhiran, G., Kuehnemuth, R., Seidel, C. A., Monzel, C., Felekyan, S.The death receptor cluster of differentiation 95 (CD95 / Fas) is an important inducer of apoptotic activity in tumor cells, but may exhibit differential cell responses (ranging from predominantly apoptosis up to proliferation) when stimulated by its CD95 ligand (CD95L/FasL). How ligand form and receptor-ligand stoichiometry shape these divergent outcomes remain unresolved. To define structural and functional determinants of CD95 signaling, we systematically compared the oligomerization and activity of native CD95L with CD95L variants, where ligand trimers were stabilized either […]
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