SEARCH · Search bioRxiv

Results for “Biophysics”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,801 records · Page 100Linked to original sources

bioRxiv2020

Two particle picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER

Structure determination of filamentous molecular complexes involves the selection of filaments from cryo-EM micrographs. The automatic selection of helical specimens is particularly difficult and thus many challenging samples with issues such as contamination or aggregation are still manually picked. Here we present two approaches for selecting filamentous complexes: one uses a trained deep neural network to identify the filaments and is integrated in SPHIRE-crYOLO, the other one, called SPHIRE-STRIPER, is based on a classical line detection approach. The advantage of the crYOLO based procedure is that it accurately performs on very challenging data sets and selects filaments with high accuracy. Although STRIPER is less precise, the user benefits from less intervention, since in contrast to crYOLO, STRIPER does not require training. We evaluate the performance of both procedures on tobacco mosaic virus and filamentous F-actin data sets to demonstrate the robustness of each method.

biophysics↗

bioRxiv2020

Mass Photometry of Membrane Proteins

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane mimetic systems at the single particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterising the particle size, sample purity and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers - in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

biophysics↗

bioRxiv2020

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

Atomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs. We show how this effect can be utilised in structural studies of nano-sized upward convex objects such as spherical or filamentous molecular assemblies deposited on a flat surface, because it causes magnification of such objects in AFM topographs. Subsequently, this enhancement effect is harnessed through contact-point based deconvolution of AFM topographs. Here, the application of this approach is demonstrated through the 3D reconstruction of the surface envelope of individual helical amyloid filaments without the need of cross-particle averaging using the contact-deconvoluted AFM topographs. Resolving the structural variations of individual macromolecular assemblies within inherently heterogeneous populations is paramount for mechanistic understanding of many biological phenomena such as amyloid toxicity and prion strains. The approach presented here will also facilitate the use of AFM for high-resolution structural studies and integrative structural biology analysis of single molecular assemblies.

biophysics↗

bioRxiv2020

Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

The DNA cytosine deaminases APOBEC3A, APOBEC3B, and APOBEC3H Haplotype I can induce mutations in cells that lead to cancer evolution. The database of cancer biomarkers in DNA repair genes (DNArCdb) identified a single nucleotide polymorphism (rs139298) of APOBEC3H Haplotype I that is involved in lung cancer1. Here, we show that this single nucleotide polymorphism causes the destabilization of APOBEC3H Haplotype I. Computational analysis suggests that the resulting K121E change affects the structure of APOBEC3H leading to active site disruption and destabilization of the RNA-mediated dimer interface. A K117E mutation in a K121E background stabilized the APOBEC3H Haplotype I, enabled biochemical study, and showed that the K121E affected catalytic activity, single-stranded DNA binding, and oligomerization on single-stranded DNA. That the destabilization of a DNA mutator would be associated with lung cancer suggests that too much mutation could result in immune recognition or death of tumor cells, suggesting that multiple APOBEC3s would not be expressed in the same tumor cells. In support of this hypothesis, stably expressed APOBEC3H Haplotype I caused a high amount of double-stranded DNA breaks in a lung cancer cell line that endogenously expressed APOBEC3B. Altogether, the data support the model that high APOBEC3 mutations in tumors are protective.

biophysics↗

bioRxiv2020

Controlled neighbor exchanges drive glassy behavior, intermittency and cell streaming in epithelial tissues

Cell neighbor exchanges are integral to tissue rearrangements in biology, including development and repair. Often these processes occur via topological T1 transitions analogous to those observed in foams, grains and colloids. However, in contrast to in non-living materials the T1 transitions in biological tissues are rate-limited and cannot occur instantaneously due to the finite time required to remodel complex structures at cell-cell junctions. Here we study how this rate-limiting process affects the mechanics and collective behavior of cells in a tissue by introducing this important biological constraint in a theoretical vertex-based model as an intrinsic single-cell property. We report in the absence of this time constraint, the tissue undergoes a motility-driven glass transition characterized by a sharp increase in the intermittency of cell-cell rearrangements. Remarkably, this glass transition disappears as T1 transitions are temporally limited. As a unique consequence of limited rearrangements, we also find that the tissue develops spaitally correlated streams of fast and slow cells, in which the fast cells organize into stream-like patterns with leader-follower interactions, and maintain optimally stable cell-cell contacts. The predictions of this work is compared with existing in-vivo experiments in Drosophila pupal development.

biophysics↗

bioRxiv2020

The within-host viral kinetics of SARS-CoV-2

In this work, we use a within-host viral dynamic model to describe the SARS-CoV-2 kinetics in host. Chest radiograph score data are used to estimate the parameters of that model. Our result shows that the basic reproductive number of SARS-CoV-2 in host growth is around 3.79. Using the same method we also estimate the basic reproductive number of MERS virus is 8.16 which is higher than SARS-CoV-2. The PRCC method is used to analyze the sensitivities of model parameters and the drug effects on virus growth are also implemented to analyze the model.

biophysics↗

bioRxiv2020

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates

The bell-shaped members of Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the somersaulting locomotion in Hydra. We applied atomic force microscopy to measure the local mechanical properties of Hydras body column and identified the existence of differential Youngs modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersault primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix (ECM) polymerization impairs the somersault movement. These results provide mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

biophysics↗

bioRxiv2020

An Inherent Structural Difference Between Serine and Threonine Phosphorylation: Phosphothreonine Prefers an Ordered, Compact, Cyclic Conformation

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted{phi} ({phi} [~] -60{degrees}) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n[->]{pi}* interaction between consecutive carbonyls, and an n[->]{sigma}* interaction between the phosphate O{gamma} lone pair and the antibonding orbital of C-H{beta} that restricts the{chi} 2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic prolines backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are{phi} ,{psi} = polyproline helix or -helix ({phi} [~] -60{degrees});{chi} 1 = g-;{chi} 2 = eclipsed C-H/O-P bonds. This structural signature is observed in diverse proteins, including the activation loops of protein kinases and protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

biophysics↗

bioRxiv2020

Mediator-Microorganism Interaction in Microbial Solar Cell: a Fluo-Electrochemical Insight

Microbial solar cells that mainly rely on the use of photosynthesic organisms are a promising alternative to photovoltaics for solar electricity production. In that way, we propose a new approach involving electrochemistry and fluorescence techniques. The coupled set-up Electro-Pulse-Amplitude-Modulation ("e-PAM") enables the simultaneous recording of the produced photocurrent and fluorescence signals from the photosynthetic chain. This methodology was validated with a suspension of green alga Chlamydomonas reinhardtii in interaction with an exogenous redox mediatior (2,6-dichlorobenzoquinone; DCBQ). The balance between photosynthetic chain events (PSII photochemical yield, quenching) and the extracted electricity can be monitored overtime. More particularly, the non photochemical quenching induced by DCBQ mirrors the photocurrent. This set-up thus helps to distinguish the electron harvesting from some side effects due to quinones in real time. It therefore paves the way for future analyses devoted to the choice of the experimental conditions (redox mediator, photosynthetic organisms...) to find the best electron extraction.

biophysics↗

bioRxiv2020

Induced Polarization in MD Simulations of the 5HT3 Receptor Channel

Ion channel proteins form water-filled nanoscale pores within lipid bilayers and their properties are dependent on the complex behavior of water in a nano-confined environment. Using the pore of the 5HT3 receptor (5HT3R) we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular Dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model, to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na+ and for Cl- ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl- whilst it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties. ToC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/971853v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@13fab4aorg.highwire.dtl.DTLVardef@106a6b0org.highwire.dtl.DTLVardef@1a97cb9org.highwire.dtl.DTLVardef@3b8dee_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Bulk-surface coupling reconciles Min-protein pattern formation in vitro and in vivo

Self-organisation of Min proteins is responsible for the spatial control of cell division in Escherichia coli, and has been studied both in vivo and in vitro. Intriguingly, the protein patterns observed in these settings differ qualitatively and quantitatively. This puzzling dichotomy has not been resolved to date. Using reconstituted proteins in laterally wide microchambers with a well-controlled height, we show that the Min protein dynamics on the membrane crucially depend on bulk gradients normal to the membrane. A theoretical analysis shows that in vitro patterns at low bulk height are driven by the same lateral oscillation mode as pole-to-pole oscillations in vivo. At larger bulk height, additional vertical oscillation modes set in, marking the transition to a qualitatively different in vitro regime. Our work qualitatively resolves the Min systems in vivo/in vitro conundrum and provides important insights on the mechanisms underlying protein patterns in bulk-surface coupled systems.

biophysics↗

bioRxiv2020

Stochastic bond dynamics facilitates alignment of malaria parasite at erythrocyte membrane upon invasion

Malaria parasites invade healthy red blood cells (RBCs) during the blood stage of the disease. Even though parasites initially adhere to RBCs with a random orientation, they need to align their apex toward the membrane in order to start the invasion process. Using hydrodynamic simulations of a RBC and parasite, where both interact through discrete stochastic bonds, we show that parasite alignment is governed by the combination of RBC membrane deformability and dynamics of adhesion bonds. The stochastic nature of bond-based interactions facilitates a diffusive-like re-orientation of the parasite at the RBC membrane, while RBC deformation aids in the establishment of apexmembrane contact through partial parasite wrapping by the membrane. This bond-based model for parasite adhesion quantitatively captures alignment times measured experimentally and demonstrates that alignment times increase drastically with increasing rigidity of the RBC membrane. Our results suggest that the alignment process is mediated simply by passive parasite adhesion.

biophysics↗

bioRxiv2020

Fourier shell correlation criteria for local resolution estimation

The resolution of three-dimensional (3D) maps obtained by electron microscopy and reconstruction is rarely constant, owing to the flexibility or compositional variability of the macromolecules of interest. Accurately estimating the local resolution within 3D reconstruction volumes is desirable for analytical purposes, and may help improve the reconstruction process itself. One approach to estimating the local resolution is to compute Fourier shell correlations (FSC) between equivalent local regions of maps computed from half datasets: the estimated local resolution is that of the Fourier shell where the FSC drops below a pre-established threshold value. Existing thresholds can lead to significant over-estimates of the local resolution because they do not take into account the low number of independent voxels in low-resolution Fourier shells typically encountered when dealing with small sub-volumes. We derive a modified FSC threshold criterion, and a simple statistical test of whether the FSC significantly exceeds it, which aims to remedy this by taking into account the bias and variance of the FSC as an estimator of signal-to-noise ratio.

biophysics↗

bioRxiv2020

Amorphous nickel titanium alloy film: a new choice for cryo electron microscopy sample preparation

Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increase successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including superior electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.

biophysics↗

bioRxiv2020

Topological defects in the nematic order of actin fibers as organization centers of Hydra morphogenesis

Animal morphogenesis arises from the complex interplay between multiple mechanical and biochemical processes with mutual feedback. Developing an effective, coarse-grained description of morphogenesis is essential for understanding how these processes are coordinated across scales to form robust, functional outcomes. Here we show that the nematic order of the supra-cellular actin fibers in regenerating Hydra defines a slowly-varying field, whose dynamics provide an effective description of the morphogenesis process. We show that topological defects in this field, which are long-lived yet display rich dynamics, act as organization centers with morphological features developing at defect sites. These observations suggest that the nematic orientation field can be considered a "mechanical morphogen" whose dynamics, in conjugation with various biochemical and mechanical signaling processes, result in the robust emergence of functional patterns during morphogenesis.

biophysics↗

bioRxiv2020

Fusion pore expansion and contraction during catecholamine release from endocrine cells

Amperometry recording reveals the exocytosis of catecholamine from individual vesicles as a sequential process, typically beginning slowly with a pre-spike foot, accelerating sharply to initiate a spike, reaching a peak, and then decaying. This complex sequence reflects the interplay between diffusion, flux through a fusion pore, and possibly dissociation from a vesicles densecore. In an effort to evaluate the impacts of these factors, a model was developed that combines diffusion with flux through a static pore. This model recapitulated the rapid phases of a spike, but generated relations between spike shape parameters that differed from experimental results. To explore the possibility of fusion pore dynamics, a transformation of amperometry current was introduced that yields fusion pore permeability divided by vesicle volume (g/V). Applying this transform to individual fusion events yielded a highly characteristic time course. g/V initially tracks the pre-spike foot and the start of the spike, increasing [~]15-fold to the peak. However, after the spike peaks, g/V unexpectedly declines and settles to a constant value that indicates the presence of a stable post-spike pore. g/V of the post-spike pore varies greatly between events, and has an average that is [~]3.5-fold below the peak value and [~]4.5-fold above the pre-spike value. The post-spike pore persists and g/V remains flat for tens of milliseconds, as long as catecholamine flux can be detected. Applying the g/V transform to rare events with two peaks revealed a stepwise increase in g/V during the second peak. The g/V transform offers an interpretation of amperometric current in terms of fusion pore dynamics and provides a new framework for analyzing the actions of proteins that alter spike shape. The stable post-spike pore conforms with predictions from lipid bilayer elasticity, and offers an explanation for previous reports of prolonged hormone retention within fusing vesicles. STATEMENT OF SIGNIFICANCEAmperometry recordings of catecholamine release from single vesicles reveal a complex waveform with distinct phases. The role of the fusion pore in this waveform is poorly understood. A model based on a static fusion pore fails to recapitulate important aspects of the waveform. A new transform of amperometric current introduced here renders fusion pore permeability in real time. This transform reveals rich dynamic behavior of the fusion pore as catecholamine leaves a vesicle. This analysis shows that fusion pore permeability rapidly increases and then decreases before settling into a stable post-spike configuration.

biophysics↗

bioRxiv2020

Real-time 3D Single Molecule Tracking

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of "locking on" to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART was applied to actively track single Atto 647N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking was further applied to single proteins and nucleic acids, directly measuring the diffusion coefficient of various lengths (99 to 1385 bp) of single DNA molecules at speeds up to 10 m2/s. In addition, 3D-SMART was able to quantify to occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

biophysics↗

bioRxiv2020

HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane domain via the C-linker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage- and cAMP-dependent mechanisms of HCN channel gating.

biophysics↗

Refine your search to explore more results.