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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 721 records · Page 40Linked to original sources

bioRxiv2017

De novo BK channel mutation causes epilepsy by regulating voltage-dependent, but not calcium-dependent, activation

Epilepsy is one of the most common neurological diseases. Here we report the first de novo mutation in the BK channel (p.N995S) that causes epilepsy in two independent families. The p.N995S mutant channel showed a markedly increased macroscopic potassium current mediated by increases in both channel open probability and channel open dwell time. Mutation p.N995S affects the voltage-activation pathway of BK channel, but does not affect the calcium sensitivity. Paxilline blocks potassium currents from both WT and mutant BK channels. We also identified two variants of unknown significance, p.E656A and p.N1159S in epilepsy patients. However, they do not affect BK channel functions, therefore, are unlikely to be a cause of disease. These results expand the BK channelopathy to a more common disease of epilepsy, suggest that the BK channel is a drug target for treatment of epilepsy, and highlight the importance of functional studies in the era of precision medicine.

biophysics↗

bioRxiv2017

High-density superresolution microscopy with an incoherent light source and a conventional epifluorescence microscope setup

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM), is an extension of single molecule localisation microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as "blinking"), detected and localised. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that STED and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution.

biophysics↗

bioRxiv2017

Dynamical Scaling And Phase Coexistence In Topologically-Constrained DNA Melting

There is a long-standing experimental observation that the melting of topologically constrained DNA, such as circular-closed plasmids, is less abrupt than that of linear molecules. This finding points to an important role of topology in the physics of DNA denaturation, which is however poorly understood. Here, we shed light on this issue by combining large-scale Brownian Dynamics simulations with an analytically solvable phenomenological Landau mean field theory. We find that the competition between melting and supercoiling leads to phase coexistence of denatured and intact phases at the single molecule level. This coexistence occurs in a wide temperature range, thereby accounting for the broadening of the transition. Finally, our simulations show an intriguing topology-dependent scaling law governing the growth of denaturation bubbles in supercoiled plasmids, which can be understood within the proposed mean field theory.

biophysics↗

bioRxiv2017

The ubiquity of directional and reciprocating motion in enzymes out of equilibrium

Molecular motors are thought to generate force and directional motion via nonequilibrium switching between energy surfaces. Because all enzymes can undergo such switching, we hypothesized that the ability to generate rotary motion and torque is not unique to highly adapted biological motor proteins, but is instead a common feature of enzymes. We used molecular dynamics simulations to compute energy surfaces for hundreds of torsions in three enzymes, adenosine kinase, protein kinase A, and HIV-1 protease, and used these energy surfaces within a kinetic model that accounts for intersurface switching and intrasurface probability flows. When substrate is out of equilibrium with product, we find computed torsion rotation rates up ~140 cycle s-1, with stall torques up to ~2 kcal mol-1 cycle-1, and power outputs up to ~50 kcal mol-1 s-1. We argue that these enzymes are instances of a general phenomenon of directional probability flows on asymmetric energy surfaces for systems out of equilibrium. Thus, we conjecture that cyclic probability fluxes, corresponding to rotations of torsions and higher-order collective variables, exist in any chiral molecule driven between states in a non-equilibrium manner; we call this the Asymmetry-Directionality conjecture. This is expected to apply as well to synthetic chiral molecules switched in a nonequilibrium manner between energy surfaces by light, redox chemistry, or catalysis.

biophysics↗

bioRxiv2017

Evidence of evolutionary selection for co-translational folding

Recent experiments and simulations have demonstrated that proteins can fold on the ribosome. However, the extent and generality of fitness effects resulting from co-translational folding remain open questions. Here we report a genome-wide analysis that uncovers evidence of evolutionary selection for co-translational folding. We describe a robust statistical approach to identify loci within genes that are both significantly enriched in slowly translated codons and evolutionarily conserved. Surprisingly, we find that domain boundaries can explain only a small fraction of these conserved loci. Instead, we propose that regions enriched in slowly translated codons are associated with co-translational folding intermediates, which may be smaller than a single domain. We show that the intermediates predicted by a native-centric model of co-translational folding account for the majority of these loci across more than 500 E. coli proteins. By making a direct connection to protein folding, this analysis provides strong evidence that many synonymous substitutions have been selected to optimize translation rates at specific locations within genes. More generally, our results indicate that kinetics, and not just thermodynamics, can significantly alter the efficiency of self-assembly in a biological context.

biophysics↗

bioRxiv2017

Iterative model-based density improvement yields better atomic structures from cryo-EM maps

Atomic models based on high-resolution density maps are the ultimate result of the cryo-EM structure determination process. Current cryo-EM model refinement procedures work with the experimental density map that remains constant throughout the process. Here, we introduce a general procedure that iteratively improves cryo-EM density maps based on prior knowledge of an atomic reference structure. The procedure optimizes contrast of cryo-EM densities by local amplitude scaling (LocScale) based on an atomic model without introducing model bias. We alternate the procedure with consecutive rounds of model refinement and tested it on four cryo-EM structures of TRPV1, {beta}-galactosidase, {gamma}-secretase and RNA polymerase III. We demonstrate that LocScale density improvement reveals previously undiscovered map features and improves the quality of atomic models. The presented approach enhances the interpretability of cryo-EM density maps and provides an implementation reminiscent of iterative density improvement as it is routinely employed in the refinement of X-ray crystallographic models.

biophysics↗

bioRxiv2017

Real-time observation of flexible domain movements in Cas9

The CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

biophysics↗

bioRxiv2017

A Conformational Checkpoint Between DNA Binding And Cleavage By CRISPR-Cas9

The Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

biophysics↗

bioRxiv2017

RRM domain of ALS/FTD-causing FUS interacts with membrane: an anchor of membraneless organelles to membranes?

526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. FUS belongs to an emerging category of proteins which are capable of forming membraneless organelles in cells via phase separation. On the other hand, eukaryotic cells contain a large network of internal membrane systems. Therefore, it is of fundamental importance to address whether membraneless organelles can interact with membranes. Here we attempted to explore this by NMR HSQC titrations of three FUS domains with gradual addition of DMPC/DHPC bicelle, which mimics the bilayer membrane. We found that both N- and C-terminal LC domains showed no significant interaction with bicelle, but its well-folded RRM domain does dynamically interact with bicelle with an interface opposite to that for binding nucleic acids including RNA and ssDNA. If this in vitro observation also occurs in cells, to interact with membrane might represent a mechanism for dynamically organizing membraneless organelles to membranes to facilitate their physiological functions.

biophysics↗

bioRxiv2017

Ultrasensitivity and Fluctuations in the Barkai-Leibler Model of Chemotaxis Receptors in Escherichia coli

A stochastic version of the Barkai-Leibler model of chemotaxis receptors in Escherichia coli is studied here with the goal of elucidating the effects of intrinsic network noise in their conformational dynamics. The model was originally proposed to explain the robust and near-perfect adaptation of E. coli observed across a wide range of spatially uniform attractant/repellent (ligand) concentrations. In the model, a receptor is either active or inactive and can stochastically switch between the two states. The enzyme CheR methylates inactive receptors while CheB demethylates active receptors and the probability for a receptor to be active depends on its level of methylation and ligand occupation. In a simple version of the model with two methylation sites per receptor (M = 2), we show rigorously, under a quasi-steady state approximation, that the mean active fraction of receptors is an ultrasensitive function of [CheR]/[CheB] in the limit of saturating receptor concentration. Hence the model shows zero-order ultrasensitivity (ZOU), similar to the classical two-state model of covalent modification studied by Goldbeter and Koshland (GK). We also find that in the limits of extremely small and extremely large ligand concentrations, the system reduces to two different two-state GK modules. A quantitative measure of the spontaneous fluctuations in activity is provided by the variance [Formula] in the active fraction, which is estimated mathematically under linear noise approximation (LNA). It is found that [Formula] peaks near the ZOU transition. The variance is a non-monotonic, but weak function of ligand concentration and a decreasing function of receptor concentration. Gillespie simulations are also performed in models with M = 2, 3 and 4. For M = 2, simulations show excellent agreement with analytical results obtained under LNA. Numerical results for M = 3 and M = 4 are qualitatively similar to our mathematical results in M = 2; while all the models show ZOU in mean activity, the variance is found to be smaller for larger M. The magnitude of receptor noise deduced from available experimental data is consistent with our predictions. A simple analysis of the downstream signaling pathway shows that this noise is large enough to affect the motility of the organism, and may have a beneficial effect on it. The response of mean receptor activity to small time-dependent changes in the external ligand concentration is computed within linear response theory, and found to have a bilobe form, in agreement with earlier experimental observations.

biophysics↗

bioRxiv2017

Single Cardiac Ventricular Myosins Change Step-Size with Loading

The cardiac myosin motor powers the beating heart by catalyzed ATPase free energy conversion to contractile work. Transgenic mouse models for heart disease express mouse -cardiac myosin heavy chain with human essential light chain (ELC) in wild type (WT), or hypertrophic cardiomyopathy linked mutant forms, A57G or E143K. Mutants modify the ELC actin binding N-terminus or C-terminus regions. Motility and single myosin mechanical characteristics show stark contrasts between the motors related to their average force, power, and displacement while all indicate the ability to down-shift ensemble step-size with increasing load. A57G and E143K consume more ATP than control WT in the presence of actin with A57G upregulating and E143K downregulating power compared with WT. Higher ATP consumption and downregulated power in E143K implies a lower unitary force. Effects on power are consistent with an A57G that impairs the ELC N-terminus actin binding and an E143K that reduces lever-arm rigidity.

biophysics↗

bioRxiv2017

Neuron Type-Specific Mechanical Regulation Of Voltage-Gated Ca2+ Channels And Excitability In Hippocampal And Trigeminal Ganglion Neurons

Increasing evidence suggests that the mechanical properties of extracellular matrix regulate central and peripheral neuronal functions. We thus investigated the CaV channels in hippocampal and trigeminal ganglion (TG) neurons cultured on substrates with different stiffness. Patch-clamp current recordings showed that stiff substrate augmented the CaV channel currents in hippocampal and TG neurons and additionally induced a leftward shift in the voltage-dependent channel activation curve in small TG neurons. Combination with using selective channel blockers revealed that substrate stiffness preferentially regulated the N-type channel current in hippocampal and medium TG neurons but the T-type channel current in small TG neurons. Current-clamp recordings further demonstrated that stiff substrate enhanced the excitability of small TG neurons, which was ablated by blocking the T-type channel. Treatment of neurons on the stiff substrate with low-dose blebbistatin reduced both the N-type channel current in hippocampal and medium TG neurons and the T-type channel current in small TG neurons to the levels in neurons on the soft substrate, whereas treatment of neurons on the soft substrate with calcium A increased both the N-type channel current in hippocampal and medium TG neurons and the T-type channel current in small TG neurons to the levels in neurons on the stiff substrate, thus consistently supporting critical involvement of actomyosin in mechanical sensing. Taken together, our results reveal neuron type-specific mechanical regulation of the Cav channels and excitability in the nervous system. Such information is useful for neural tissue engineering and regeneration.

biophysics↗

bioRxiv2017

Landmark Kernel tICA For Conformational Dynamics

Molecular dynamics simulations of biomolecules produce a very high dimensional time-series dataset. Performing analysis necessarily involves projection onto a lower dimensional space. A priori selection of projection coordinates requires (perhaps unavailable) prior information or intuition about the system. At best, such a projection can only confirm the intuition. At worst, a poor projection can obscure new features of the system absent from the intuition. Previous statistical methods such a time-structure based independent component analysis (tICA) and Markov state modeling (MSMs) have offered relatively unbiased means of projecting conformations onto coordinates or state labels, respectively. These analyses are underpinned by the propagator formalism and the assumption that slow dynamics are biologically interesting. Although arising from the same mathematics, tICA and MSMs have different strengths and weaknesses. We introduce a unifying method which we term \"landmark kernel tICA\" (lktICA) which uses a variant of the Nystrom kernel approximation to permit approximate non-linear solutions to the tICA problem. We show that lktICA is equivalent to MSMs with \"soft\" states. We demonstrate the advantages of this united method by finding improved projections of (a) a 1D potential surface (b) a peptide folding trajectory and (c) an ion channel conformational change.

biophysics↗

bioRxiv2017

Revealing The Cell-Material Interface With Nanometer Resolution By FIB-SEM

The interface between biological cells and non-biological surfaces profoundly influences cellular activities, chronic tissue responses, and ultimately the success of medical implants. Materials in contact with cells can be plastics, metal, ceramics or other synthetic materials, and their surfaces vary widely in chemical compositions, stiffness, topography and levels of roughness. To understand the molecular mechanism of how cells and tissues respond to different materials, it is of critical importance to directly visualize the cell-material interface at the relevant length scale of nanometers. Conventional ultrastructural analysis by transmission electron microscopy (TEM) often requires substrate removal before microtome sectioning, which is not only challenging for most substrates but also can cause structural distortions of the interface. Here, we present a new method for in situ examination of the cell-to-material interface at any desired cellular location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM). This method involves a thin-layer plastification procedure that preserves adherent cells as well as enhances the contrast of biological specimen. We demonstrate that this unique procedure allows the visualization of cell-to-material interface and intracellular structures with 10nm resolution, compatible with a variety of materials and surface topographies, and capable of volume and multi-directional imaging. We expect that this method will be very useful for studies of cell-to-material interactions and also suitable for in vivo studies such as examining osteoblast adhesion and new bone formation in response to titanium implants.

biophysics↗

bioRxiv2017

Intracellular Passage Of Na+ In An Active State G Protein Coupled Receptor

Playing a central role in cell signalling, GPCRs have evolved into the largest superfamily of membrane proteins and form the majority of drug targets in humans. How extracellular agonist binding triggers the activation of GPCRs and associated intracellular effector proteins remains, however, poorly understood. High resolution structural studies have recently revealed that inactive class-A GPCRs harbour a conserved binding site for Na+ ions in the centre of their transmembrane domain, accessible from the extracellular space. Here, we show that the opening of a conserved hydrated channel in the activated state receptors allows the Na+ ion to egress from its binding site into the cytosol. Coupled with protonation changes, this ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. We propose that Na+ ion exchange with the cytosol is a key step in GPCR activation, which locks receptors in long-lived active-state conformations.

biophysics↗

bioRxiv2017

Thermodynamic Projection Of The Antibody Interaction Network: The Binding Fountain Energy Landscape

The complexity of the adaptive immune system in humans is comparable to that of the central nervous system in terms of cell numbers, cellular diversity and the network of interactions between components. While the application of molecular biological methods and bioinformatics has brought about an ever deepening and sharpening static description of the molecular and cellular components of the system, a unifying theoretical understanding of the laws governing the dynamics of the system is still lacking.\n\nWe have recently developed a quantitative model for the description of antibody homeostasis as defined by the dimensions of antigen concentration, antigen-antibody interaction affinity and antibody concentration. In this paper we develop the concept of a novel thermodynamic representation of multiple molecular interactions in a system, the fountain energy landscape of binding. We show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network projected onto an energy landscape defined by conformational entropy and free energy of binding. We demonstrate that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the binding fountain energy landscape concept allows a systems biological, thermodynamic perception of the functioning of the clonal humoral immune system.

biophysics↗

bioRxiv2017

Transcription factor clusters regulate genes in eukaryotic cells

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

biophysics↗

bioRxiv2017

Formation Of Dominant Mode By Evolution In Biological Systems

A reduction in high-dimensional phenotypic states to a few degrees of freedom is essential to understand biological systems. One possible origin of such a reduction (as recently discussed) is the steady growth of cells that constrains each components replication rate. Here, in contrast, our aim is to investigate consequences of evolutionary robustness, which is shown to cause a stronger dimensional reduction in possible phenotypic changes in response to a variety of environmental conditions. First, we examined global protein expression changes in Escherichia coli after various environmental perturbations. Remarkably, they were proportional across components, across different types of environmental conditions, while the proportion coefficient corresponded to the change in growth rate. Because such global proportionality is not generic to all systems under a condition of steady growth, a new conceptual framework is then needed. We hypothesized that such proportionality is a result of evolution. To test this hypothesis, we analyzed a cell model--with a huge number of components, that reproduces itself via a catalytic reaction network--and confirmed that common proportionality in the concentrations of all components is shaped through evolutionary processes to maximize cell growth (and therefore fitness) under a given environmental condition. Furthermore, we found that the changes in concentration across all components in response to environmental and evolutionary changes are constrained to the changes along a one-dimensional major axis within a huge-dimensional state space. On the basis of these observations, we propose a theory in which high-dimensional phenotypic changes after evolution are constrained to the points near a one-dimensional major axis that correlates with the growth rate, to achieve both evolutionary robustness and plasticity. By formulating this proposition in terms of dynamical systems, broad experimental and numerical results on phenotypic changes caused by evolution and adaptation are coherently explained.\n\nSummaryCells generally consist of thousands of components whose abundance levels change through adaptation and evolution. Accordingly, each steady cell state can be represented as a point in a high-dimensional space of component concentrations. In the context of equilibrium statistical thermodynamics, even though the state space is high-dimensional, macroscopic description only by a few degrees of freedom is possible for equilibrium systems; however, such characterization by a few degrees of freedom has not yet been achieved for cell systems. Given that they are not in equilibrium, we need some other constraint to be imposed. Here, by restricting our focus to a cellular state with steady growth that is achieved after evolution,we examine how the expression levels of its several components change under different environmental conditions. On the basis of analysis of protein expression levels in recent bacterial experiments as well as the results of simulations using a toy cell model consisting of thousands of components that are reproduced by catalytic reactions, we found that adaptation and evolutionary paths in high-dimensional state space are constrained to changes along a one-dimensional curve, representing a major axis for all the observed changes. Moreover, this one-dimensional structure emerges only after evolution and is not applicable to any system showing steady growth. This curve is determined by the growth rate of a cell, and thus it is possible to describe an evolved system by means of a growth rate function. All the observed results are consistent with the hypothesis that changes in high-dimensional states are nearly confined to the major axis in response to environmental, evolutionary, and stochastic perturbations. This description opens up the possibility to characterize a cell state as a macroscopic growth rate, as is the case for the thermodynamic potential. This approach can provide estimates of which phenotypic changes are theoretically more evolvable, as predicted simply from their observed environmental responses.

biophysics↗