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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Cryo-EM structure of the polycystin 2-l1 ion channel

We report the near atomic resolution (3.1 [A]) of the polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1.

biophysics↗

bioRxiv2018

Non-productive Binding Modes as a Prominent Feature of Aβ1-40 Fiber Elongation: Insights from Molecular Dynamics Simulation

AbstractAmyloid formation has been implicated in a number of neurodegenerative diseases. The elongation of amyloid fibers is thermodynamically strongly favorable but kinetic traps exist where the incoming monomer binds in an incompatible conformation that blocks further elongation. Unfortunately, this process is difficult to follow experimentally at the atomic level. It is also too complex to simulate in full detail and thus so far has been explored either through coarse-grained simulations, which may miss many important interactions, or full atomic simulations in which the incoming peptide is constrained to be near the ideal fiber geometry. Here we use an alternate approach starting from a docked complex in which the monomer is from an experimental NMR structure of one of the major conformations in the unbound ensemble, a largely unstructured peptide with the central hydrophobic region in a 310 helix. A 1000 ns full atomic simulation in explicit solvent shows the formation of a metastable intermediate by sequential, concerted movements of both the fiber and monomer. A Markov state model shows the unfolded monomer is trapped at the end of the fiber in a set of interconverting anti-parallel {beta}-hairpin conformations. The simulation here may serve as a model for the binding of other non-{beta}-sheet conformations to amyloid fibers.

biophysics↗

bioRxiv2018

Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not found in the isolated S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single ATP turnover kinetics. A two-headed myosin fragment exhibits distinct auto-inhibited ATP turnover kinetics compared to a single-headed fragment. Mavacamten enhanced this auto-inhibition. It also enhanced auto-inhibition of ADP release. Furthermore, actin changes the structure of the auto-inhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.\n\nSignificance StatementSmall-molecule allosteric effectors designed to target and modulate striated and smooth myosin isoforms for the treatment of disease show promise in preclinical and clinical trials. Beta-cardiac myosin is an especially important target, as heart disease remains a primary cause of death in the U.S. One prevalent type of heart disease is hypertrophic cardiomyopathy (HCM), which is hypothesized to result from dysregulated force generation by cardiac myosin. Mavacamten is a potent cardiac myosin ATPase activity inhibitor that improves cardiac output in HCM animal models. Our results show that mavacamten selectively stabilizes a two-head dependent, auto-inhibited state of cardiac myosin in solution. The kinetics and energetics of this state are consistent with the auto-inhibited super-relaxed state, previously only observed in intact sarcomeres.

biophysics↗

bioRxiv2018

Nanoscale spatio-temporal diffusion modes measured by simultaneous confocal and STED imaging

The diffusion dynamics in the cellular plasma membrane provides crucial insights into the molecular interactions, organization and bioactivity. Fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (STED-FCS) measures such dynamics with high spatial and temporal resolution and reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method; line interleaved excitation scanning STED-FCS (LIESS-FCS) which discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatio-temporal organization of GPI-anchored proteins in the plasma membrane of live cells which interestingly show multiple diffusion modes at different spatial positions.

biophysics↗

bioRxiv2018

Kinetic analysis methods applied to single motor protein trajectories

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard to resolve, as they are masked by thermal noise. In such cases, details of the mechanochemistry can nonetheless be recovered by analyzing the fluctuations in the recorded traces. Here, we expand upon existing statistical analysis methods, providing two new avenues to extract the motor step size, the effective number of rate-limiting chemical states per translocation step, and the compliance of the link between the motor position and the probe particle. We first demonstrate the power and limitations of these methods using simulated molecular motor trajectories, and we then apply these methods to experimental data of kinesin, the bacterial flagellar motor, and F1-ATPase.

biophysics↗

bioRxiv2018

Reducing effects of particle adsorption to the air-water interface in cryoEM

Most protein particles prepared in vitreous ice for single particle cryo-electron microscopy are adsorbed to air-water or substrate-water interfaces, potentially causing particles to adopt preferred orientations. Using the Spotiton robot and nanowire grids, we can significantly reduce air-water interface issues by decreasing the dwell time of particles in thin liquid films. We demonstrate this by using single particle cryoEM and cryoET on three biological samples.

biophysics↗

bioRxiv2018

Simulations and Electrostatic Analysis Suggest an Active Role for DNA Conformational Changes During Genome Packaging by Bacteriophages

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens, as well as dsDNA bacteriophages - viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is, instead, an active component of the machinery. Computational studies on dsDNA in the channel of viral portal proteins reported here reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer (\"stretched\") in regions of high negative electrostatic potential, and shorter (\"scrunched\") in regions of high positive potential. These results suggest a mechanism that couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal and dsDNA work synergistically to promote genome packaging.

biophysics↗

bioRxiv2018

Dynamic and Structural Modeling of the Specificity in Protein-DNA Interactions Guided by Binding Assay and Structure Data

How transcription factors (TFs) recognize their DNA sequences is often investigated complementarily by high-throughput protein binding assays and by structural biology experiments. The former quantifies the specificity of TF binding sites for numerous DNA sequences, often represented as the position-weight-matrix (PWM). The latter provides mechanistic insights into the interactions via the protein-DNA complex structures. However, these two types of data are not readily integrated. Here, we propose and test a new modeling method that incorporates the PWM with complex structure data. Based on pre-tuned coarse-grained models for proteins and DNAs, we model the specific protein-DNA interactions, PWMcos, in terms of an orientation-dependent potential function, which enables us to perform molecular dynamics simulations at unprecedentedly large scales. We show that the PWMcos model reproduces subtle specificity in the protein-DNA recognition. During the target search in genomic sequences, TF moves on highly rugged landscapes and occasionally flips on DNA depending on the sequence. The TATA-binding protein exhibits two remarkably distinct binding modes, of which frequencies differ between TATA-containing and TATA-less promoters. The PWMcos is general and can be applied to any protein-DNA interactions given their PWMs and complex structure data are available.

biophysics↗

bioRxiv2018

Conformational dynamics of a photosynthetic light-harvesting complex in native thylakoid membranes

Photosynthetic light-harvesting complexes of higher plants, moss and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr Light-Harvesting Complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states. STATEMENT OF SIGNIFICANCEPhotosynthetic Light-Harvesting Complexes undergo dynamic conformational transitions that regulate the capacity of the light-harvesting antenna. We demonstrate the application of solid-state (ss)NMR spectroscopy to investigate the structural dynamics of LHCII, the most abundant LHC complex of plants and algae, in native membranes. Selective dynamic protein and pigment residues are identified that are putative sites for a conformational switch.

biophysics↗

bioRxiv2018

The Role of Cytoplasmic Interactions in the Collective Polarization of Tissues and its Interplay with Cellular Geometry

Planar cell polarity (PCP), the ability of a tissue to polarize coherently over multicellular length scales, provides the directional information that guides a multitude of developmental processes at cellular and tissue levels. While it is manifest that cells utilize both intra-cellular and intercellular mechanisms, how they couple together to produce the collective response remains an active area of investigation. Exploring a phenomeno-logical reaction-diffusion model, we predict a crucial, and novel, role for cytoplasmic interactions in the large-scale correlations of cell polarities. We demonstrate that finite-range (i.e. nonlocal) cytoplasmic interactions are necessary and sufficient for the robust and long-range polarization of tissues -- even in the absence of global cues -- and are essential to the faithful detection of weak directional signals. Strikingly, our model re-capitulates an observed influence of anisotropic tissue geometries on the orientation of polarity. In order to facilitate a conversation between theory and experiments, we compare five distinct classes of in silico mutants with experimental observations. Within this context, we propose quantitative measures that can guide the search for the participant molecular components, and the identification of their roles in the collective polarization of tissues.

biophysics↗

bioRxiv2018

Temperature-dependent estimation of Gibbs energies using an updated group contribution method

Reaction equilibrium constants determine the mass action ratios necessary to drive flux through metabolic pathways. Group contribution methods offer a way to estimate reaction equilibrium constants at wide coverage across the metabolic network. Here, we present an updated group contribution method with: 1) additional curated thermodynamic data used in fitting; and 2) capabilities to calculate equilibrium constants as a function of temperature. We first collected and curated aqueous thermo-dynamic data, including reaction equilibrium constants, enthalpies of reaction, Gibbs free energies of formation, enthalpies of formation, entropies change of formation of compounds, and proton and metal ion binding constants. We further estimated magnesium binding constants for 618 compounds using a linear regression model validated against measured data. Next, we formulated the calculation of equilibrium constants as a function of temperature and calculated necessary parameters, including standard entropy change of formation ({Delta}f S{circ}) and standard entropy change of reaction ({Delta}rS{circ}), using a model based on molecular properties. The median absolute errors in estimating {Delta}f S{circ} and {Delta}rS{circ} were 0.010 kJ/K/mol and 0.018 kJ/K/mol, respectively. The efforts here fill in gaps for thermodynamic calculations under various conditions, specifically different temperatures and metal ion concentrations. These results support the study of thermodynamic driving forces underlying the metabolic function of organisms living under diverse conditions.

biophysics↗

bioRxiv2018

A simple and robust procedure for preparing graphene-oxide cryo-EM grids

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

biophysics↗

bioRxiv2018

Standardized microgel beads as elastic cell mechanical probes

Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acylamide (PAAm) microgel beads mimicking biological cells. We produced mono-disperse beads at rates of 20 - 60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors.\n\nSignificance StatementOften vastly different cell mechanical properties are reported even for the same cell type when employing different measurement techniques. This discrepancy shows the urgent need for standardized calibration particles to cross-compare and validate techniques. Microgel beads can serve this purpose, but they have to fulfil specific requirements such as homogeneity, sizes and elasticities in the range of the cells, and they have to provide comparable results independent of the method applied. Here we demonstrate the standardized production of polyacrylamide microgel beads with all the features an elastic cell-mimic should have. These can not only be used as method calibration particles, but can also serve as cell-scale sensors to quantify normal and shear stresses exerted by other cells and inside tissues, enabling many new applications.

biophysics↗

bioRxiv2018

Cell membranes resist flow

The fluid-mosaic model posits a liquid-like plasma membrane, which can flow in response to tension gradients. It is widely assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds. This conjectured signaling mechanism has been invoked to explain how cells coordinate changes in shape, motility, and vesicle fusion, but the underlying propagation has never been observed. Here we show that propagation of membrane tension occurs quickly in cell-attached blebs, but is largely suppressed in intact cells. The failure of tension to propagate in cells is explained by a fluid dynamical model that incorporates the flow resistance from cytoskeleton-bound transmembrane proteins. In primary endothelial cells, local increases in membrane tension lead only to local activation of mechanosensitive ion channels and to local vesicle fusion. Thus membrane tension is not a mediator of long-range intra-cellular signaling, but local variations in tension mediate distinct processes in sub-cellular domains.

biophysics↗

bioRxiv2018

Entropic Forces Drive Clustering and Spatial Localization of Influenza A M2 During Viral Budding

The influenza A matrix 2 (M2) transmembrane protein facilitates virion release from the infected host cell. In particular, M2 plays a role in the induction of membrane curvature and/or in the scission process whereby the envelope is cut upon virion release. Here we show using coarse-grained computer simulations that various M2 assembly geometries emerge due to an entropic driving force, resulting in compact clusters or linearly extended aggregates as a direct consequence of the lateral membrane stresses. Conditions under which these protein assemblies will cause the lipid membrane to curve are explored and we predict that a critical cluster size is required for this to happen. We go on to demonstrate that under the stress conditions taking place in the cellular membrane as it undergoes large-scale membrane remodeling, the M2 protein will in principle be able to both contribute to curvature induction and sense curvature in order to line up in manifolds where local membrane line tension is high. M2 is found to exhibit linactant behavior in liquid-disordered/liquid-ordered phase-separated lipid mixtures and to be excluded from the liquid-ordered phase, in near-quantitative agreement with experimental observations. Our findings support a role for M2 in membrane remodeling during influenza viral budding both as an inducer and a sensor of membrane curvature, and they suggest a mechanism by which localization of M2 can occur as the virion assembles and releases from the host cell, independent of how the membrane curvature is produced.\n\nSIGNIFICANCE STATEMENTFor influenza virus to release from the infected host cell, controlled viral budding must finalize with membrane scission of the viral envelope. Curiously, influenza carries its own protein, M2, which can sever the membrane of the constricted budding neck. Here we elucidate the physical mechanism of clustering and spatial localization of the M2 scission proteins through a combined computational and experimental approach. Our results provide fundamental insights into how M2 clustering and localization interplays with membrane curvature, membrane lateral stresses, and lipid bilayer phase behavior during viral budding in order to contribute to virion release.

biophysics↗

bioRxiv2018

Signal-to-Noise ratio made easy: A tool to assess your confocal performance

Confocal microscopy is used today on a daily basis in life science labs. This \"routine\" technique contributes to the progress of scientific projects across many fields by revealing structural details and molecular localization, but researchers need to be aware that detection efficiency and emission light path performance is of major influence in the confocal image quality. By design, a large portion of the signal is discarded in confocal imaging, leading to a decreased signal-to-noise ratio (SNR) which in turn limits resolution. A well-aligned system and high performance detectors are needed in order to generate an image of best quality. However, a convenient method to address system status and performance on the emission side is still lacking. Here, we present a complete method to assess microscope and emission light path performance in terms of SNR, with a comprehensive protocol alongside NoiSee, an easy-to-use macro for Fiji (available via the corresponding update site). We used this method to compare several confocal systems in our facility on biological samples under typical imaging conditions. Our method reveals differences in microscope performance and highlights the various detector types used (multialkali photomultiplier tube (PMT), gallium arsenide phosphide (GaAsP) PMT, and Hybrid detector). Altogether, our method will provide useful information to research groups and facilities to diagnose their confocal microscopes.

biophysics↗

bioRxiv2018

Geometry-dependent instabilities in electrically excitable tissues

Little is known about how individual cells sense the macroscopic geometry of their tissue environment. Here we explore whether long-range electrical signaling can convey information on tissue geometry to influence electrical dynamics of individual cells. First, we studied an engineered electrically excitable cell line where all voltage-gated channels were well characterized. Cells grown in patterned islands of different shapes showed remarkably diverse firing patterns under otherwise identical conditions, including regular spiking, period-doubling alternans, and arrhythmic firing. A Hodgkin-Huxley numerical model quantitatively reproduced these effects, showing how the macroscopic geometry affected the single-cell electrophysiology via the influence of gap junction-mediated electrical coupling. Qualitatively similar geometry dependent dynamics were experimentally observed in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. The cardiac results urge caution in translating observations of arrhythmia in vitro to predictions in vivo where the tissue geometry is very different. We present simulation results and scaling arguments which explore how to extrapolate electrophysiological measurements between tissues with different geometries and different gap junction couplings.

biophysics↗

bioRxiv2018

Functional Pathways of Biomolecules Retrieved from Single-particle Snapshots

We present a new approach to determining the conformational changes associated with biological function, and demonstrate its capabilities in the context of experimental single-particle cryo-EM snapshots of ryanodine receptor (RyR1), a Ca2+-channel involved in skeletal muscle excitation/contraction coupling. These results include the detailed conformational motions associated with functional paths including transitions between energy landscapes. The functional motions differ substantially from those inferred from discrete structures, shedding new light on the gating mechanism in RyR1. The differences include the conformationally active structural domains, the nature, sequence, and extent of conformational motions involved in function, and the way allosteric signals are transduced within and between domains. The approach is general, and applicable to a wide range of systems and processes.

biophysics↗