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bioRxiv2020

Developing a Coarse-Grained Model for Bacterial Cell Walls and Evaluating Mechanical Properties and Free Energy Barriers

The bacterial cell envelope of Gram-negative bacteria is a complex biological barrier with multiple layers consisting of the inner membrane, periplasm of peptidoglycan and the outer membrane with lipopolysaccharides (LPS). With rising antimicrobial resistance there is increasing interest in understanding interactions of small molecules with the cell membrane to aid in the development of novel drug molecules. Hence suitable representations of the bacterial membrane are required to carry out meaningful molecular dynamics simulations. Given the complexity of the cell envelope, fully atomistic descriptions of the cell membrane with explicit solvent are computationally prohibitive, allowing limited sampling with small system sizes. However coarse-grained (CG) models such as MARTINI allow one to study phenomena at physiologically relevant length and time scales. Although MARTINI models for lipids and the LPS are available in literature, a suitable CG model of peptidoglycan is lacking. In this manuscript we develop a CG model of the peptidoglycan network within the MARTINI framework using an all-atom model developed by Gumbart et al. 1. The model is parametrized to reproduce the structural properties of the glycan strands, such as the end-to-end distance, equilibrium angle between adjacent peptides along the strands and area per disaccharide. Mechanical properties such as the area compressibility and the bending modulus are accurately reproduced. While developing novel antibiotics it is important to assess barrier properties of the peptidogylcan network. We evaluate and compare the free energy of insertion for a thymol molecule using umbrella sampling on both the MARTINI and all-atom peptidoglycan models. The insertion free energy was found to be less than kBT for both the MARTINI and all-atom models. Additional restraint free simulations reveal rapid translocation of thymol across peptidogylcan. We expect that the proposed MARTINI model for peptidoglycan will be useful in understanding phenomena associated with bacterial cell walls at larger length and time scales, thereby overcoming the current limitations of all-atom models.

biophysics↗

bioRxiv2020

Activation of Macrophages by CpG DNA and LPS: an FTIR Spectroscopic Study

The innate immune response triggered by CpG DNA can improve host survival following pathogen challenge. Whether CpG ODN-mediated immune activation leads to global molecular changes in cells that are detectable by FTIR spectroscopy is currently unknown. Here, we used Attenuated Total Reflectance (ATR) Fourier Transform Infrared (FTIR) spectroscopy to monitor the molecular changes in murine macrophage RAW 264.7 cells upon activation with CpG DNA and lipopolysaccharide (LPS). By PCA analysis, we identified the sources of variation to follow with detailed spectral analysis. CpG DNA and LPS treatment increase the total nucleic acid concentration from the early periods post-activation, and DNA synthesis follows RNA synthesis. RNA-specific peak shows the activation state of macrophages in early periods post-treatment. CpG DNA and LPS result in an initial rapid increase in the total protein concentration, leveling off two hours post-activation. Both activated groups increase the concentration of fatty acids, triglycerides, and cholesterol, pointing out to a shared synthesis pathway and de novo lipogenesis. This study, for the first time, demonstrates the use of FTIR spectroscopy as an independent modality to monitor the activation dynamics of murine macrophages upon activation with CpG DNA and LPS.

biophysics↗

bioRxiv2020

A Piezo1 open state reveals a multi-fenestrated ion permeation pathway

Force-sensing Piezo channels are essential to many aspects of vertebrate physiology. Activation of Piezo1 is facilitated by the presence of negative membrane lipids in the inner leaflet, such as phosphatidylinositol-4,5-bisphosphate (PIP2). Here, to study how Piezo1 opens, we performed molecular dynamics simulations of Piezo1 in membranes flattened by the periodic boundary effect and with or without PIP2 lipids. The Piezo1 pore spontaneously opens in the asymmetrical bilayer but not in the symmetric membrane or when PIP2 lipids are neutralized. Electrophysiological characterization of putative PIP2-interacting Piezo1 residues suggests the contribution of multiple PIP2 binding sites. Our Piezo1 open state recapitulates ionic selectivity, unitary conductance and mutant phenotypes obtained from numerous experimental studies. Tracking ion diffusion through the open pore reveals the presence of intracellular and extracellular fenestrations, delineating a multi-fenestrated permeation pathway. This open state sheds light on the mechanisms of lipid modulation, permeation, and selectivity in a Piezo channel.

biophysics↗

bioRxiv2020

The Particle Filter Method to Integrate High-Speed Atomic Force Microscopy Measurement with Biomolecular Simulations

The high-speed atomic force microscopy (HS-AFM) can observe structural dynamics of biomolecules at single-molecule level in real time near physiological condition, but its spatiotemporal resolution is limited. Complementarily, molecular dynamics (MD) simulations have higher spatiotemporal resolutions albeit with some artifact. Here, in order to integrate the HS-AFM data and coarse-grained (CG)-MD simulations, we develop a particle filter method, one of the sequential Bayesian data assimilation approaches. We tested the method in a twin experiment. We first made a reference HS-AFM movie from a CG-MD trajectory of a test molecule, a nucleosome, which serves as an "experimental measurement". Then, we performed the particle filter simulation with 512 particles that captured large-scale nucleosome structural dynamics compatible with the AFM movie. Comparing the particle filter simulations with 8 - 8192 particles, we found that the use of more particles consistently results in larger likelihood for the whole AFM movie. By comparing the likelihoods from different ionic concentrations and from different timescales, we found that the "true" concentration and timescale can be inferred as the largest likelihood of the whole AFM movie, but not that of each AFM image. The particle filter method provides a general approach to integrate the HS-AFM data with MD simulations.

biophysics↗

bioRxiv2020

The Glycosphingolipid GM3 Modulates Conformational Dynamics of the Glucagon Receptor

The extracellular domain (ECD) of Class B1 G-protein coupled receptors (GPCRs) plays a central role in signal transduction and is uniquely positioned to sense both the extracellular and membrane environments. Whilst recent studies suggest a role for membrane lipids in the modulation of Class A and Class F GPCR signalling properties, little is known about the effect of lipids on Class B1 receptors. In this study, we employed multiscale molecular dynamics (MD) simulations to access the dynamics of the glucagon receptor (GCGR) ECD in the presence of native-like membrane bilayers. Simulations showed that the ECD could move about a hinge region formed by residues Q122-E126 to adopt both closed and open conformations relative to the TMD. ECD movements were modulated by binding of the glycosphingolipid GM3. These large-scale fluctuations in ECD conformation that may affect the ligand binding and receptor activation properties. We also identify a unique PIP2 interaction profile near ICL2/TM3 at the G-protein coupling interface, suggesting a mechanism of engaging G-proteins which may have a distinct dependence on PIP2 compared to Class A GPCRs. Given the structural conservation of Class B1 GPCRs, the modulatory effects of GM3 and PIP2 on GCGR may be conserved across these receptors, offering new insights into potential therapeutic targeting. Statement of SignificanceThe role of lipids in regulation of Class B GPCRs remains elusive, despite recent structural advances. In this study, multi-scale molecular dynamics simulations are used to evaluate lipid interactions with the glucagon receptor, a Class B1 GPCR. We find that the glycosphingolipid GM3 binds to the glucagon receptor extracellular domain (ECD), modulating the dynamics of the ECD and promoting movement away from the transmembrane domain. We also identify a unique PIP2 interaction fingerprint in a region known to be important for bridging G-protein coupling in Class A GPCRs. Thus, this study provides molecular insight into the behaviour of the glucagon receptor in a complex lipid bilayer environment which may aid understanding of glucagon receptor signalling properties.

biophysics↗

bioRxiv2020

Molecular resolution imaging by post-labeling expansion single molecule localization microscopy (Ex-SMLM)

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

biophysics↗

bioRxiv2020

Protein dynamics enables phosphorylation of buried residues in Cdk2/Cyclin A-bound p27

Proteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of proteins and plays key roles in the regulation of many biological processes. The finding that many phosphorylated residues are not solvent exposed in the unphosphorylated state opens several questions for understanding the mechanism that underlies phosphorylation and how phosphorylation may affect protein structures. First, since kinases need access to the phosphorylated residue, how do such buried residues become modified? Second, once phosphorylated, what are the structural effects of phosphorylation of buried residues and do they lead to changed conformational dynamics. We have used the ternary complex between p27, Cdk2 and Cyclin A to study these questions using enhanced sampling molecular dynamics simulations. In line with previous NMR and single-molecule fluorescence experiments we observe transient exposure of Tyr88 in p27, even in its unphosphorylated state. Once Tyr88 is phosphorylated, we observe a coupling to a second site, thus making Tyr74 more easily exposed, and thereby the target for a second phosphorylation step. Our observations provide atomic details on how protein dynamics plays a role in modulating multi-site phosphorylation in p27, thus supplementing previous experimental observations. More generally, we discuss how the observed phenomenon of transient exposure of buried residues may play a more general role in regulating protein function. Significance StatementProtein phosphorylation is a common post-translation modification and is carried out by kinases. While many phosphorylation sites are located in disordered regions of proteins or in loops, a surprisingly large number of modification sites are buried inside folded domains. This observation led us to ask the question of how kinases gain access to such buried residues. We used the complex between p27, a regulator of cell cycle progression, and Cyclin-dependent kinase 2/Cyclin A to study this problem. We hypothesized that transient exposure of buried tyrosines in p27 to the solvent would make them accessible to kinases, explaining how buried residues get modified. We provide an atomic-level description of these dynamic processes revealing how protein dynamics plays a role in regulation.

biophysics↗

bioRxiv2020

Architecture and subunit dynamics of the mitochondrial TIM9·10·12 chaperone

The mitochondrial Tim chaperones are responsible for the transport of membrane proteins across the inter-membrane space to the inner and outer mitochondrial membranes. TIM9{middle dot}10, a hexameric 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9{middle dot}10{middle dot}12 complex is the anchor point at the inner-membrane insertase complex TIM22. The subunit composition of the TIM9{middle dot}10{middle dot}12 complex remains debated. Joint NMR, small-angle X-ray scattering and MD simulation data allow us to derive a structural model of the TIM9{middle dot}10{middle dot}12 assembly, which has a 2:3:1 stoichiometry (Tim9:Tim10:Tim12). We find that both TIM9{middle dot}10 and TIM9{middle dot}10{middle dot}12 hexamers are in a dynamic equilibrium with their constituent subunits, exchanging on a minutes time scale. Residue-resolved NMR data establish that the subunits exhibit large conformational dynamics: when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short marginally stable -helices are formed, and these are fully stabilized only upon formation of the mature hexameric chaperone. We propose that the continuous subunit exchange is a means of mitochondria to control their level of inter-membrane space chaperones, and thus rapidly adapt to the cellular state.

biophysics↗

bioRxiv2020

Carbon ion dosimetry on a fluorescent nuclear track detector using widefield microscopy

Fluorescent nuclear track detectors (FNTD) are solid-state dosimeters used in a wide range of dosimetric and biomedical applications in research worldwide. FNTDs are a core but currently underutilized dosimetry tool in the field of radiation biology which are inherently capable of visualizing the tracks of ions used in hadron therapy. The ions that traverse the FNTD deposit their energy according to their linear energy transfer (LET) and transform colour centres to form trackspots around their trajectory. These trackspots have fluorescent properties which can be visualized by fluorescence microscopy enabling a well-defined dosimetric readout with a spatial component indicating the trajectory of individual ions. The current method used to analyse the FNTDs is laser scanning confocal microscopy (LSM). LSM enables a precise localization of track spots in x, y and z however due to the scanning of the laser spot across the sample, requires a long time for large samples. This body of work conclusively shows for the first time that the readout of the trackspots present after 0.5 Gy carbon ion irradiation in the FNTD can be captured with a widefield microscope (WF). The WF readout of the FNTD is a factor [~]10 faster, for an area 2.97 times the size making the method nearly a factor 19 faster in track acquisition than LSM. The dramatic decrease in image acquisition time in WF presents an alternative to LSM in FNTD workflows which are limited by time, such as biomedical sensors which combine FNTDs with live cell imaging.

biophysics↗

bioRxiv2020

Consistency of superb microvascular imaging and contrast enhanced ultrasonography in detecting intraplaque neovascularization: a meta-analysis

This meta-analysis aimed to identify the consistency of superb microvascular imaging(SMI) and contrast-enhanced ultrasonography(CEUS) in detecting intraplaque neovascularization(IPN). We searched PubMed, Web of Science, Cochrane Library, CISCOM, and CBM databases without language restrictions. Meta-analysis was conducted using STATA version 15.1 software. We calculated the pooled Kappa index. Ten studies that met all inclusion criteria were included in this meta-analysis. A total of 608 carotid plaques were assessed through both SMI and CEUS. The pooled summary Kappa index was 0.743(95 % CI=0.696-0.790) with statistical significance(z= 31.14, p<0.01). We found no evidence for publication bias (t=-1.21, p=0.261). Our meta-analysis indicates that SMI and CEUS display a good consistency in detecting IPN of carotid plaque, that is to say SMI ultrasound maybe a promising alternative to CEUS for detecting IPN of carotid plaque.

biophysics↗

bioRxiv2020

Adaptive FDR thresholding of Fourier shell correlation for resolution estimation of cryo-EM maps

Fourier shell correlation (FSC) has become a standard quantity for resolution estimation in electron cryo-microscopy. However, the resolution determination step is still subjective and not fully automated as it involves a series of map interventions before FSC computation and includes the selection of a common threshold. Here, we apply the statistical methods of permutation sampling and false discovery rate (FDR) control to the resolution-dependent correlation measure. The approach allows fully automated and mask-free resolution determination based on adaptive thresholding of FSC curves. We demonstrate the applicability for global, local and directional resolution estimation and show that the developed criterion termed FDR-FSC gives realistic resolution estimates based on a statistical significance criterion while eliminating the need of any map manipulations. The algorithms are implemented in a user-friendly GUI based software tool termed SPoC (https://github.com/MaximilianBeckers/SPOC).

biophysics↗

bioRxiv2020

Anomalous Diffusion Characterization by Fourier-Transform FRAP with Patterned Illumination

Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination is theorized and demonstrated for quantitatively evaluating normal and anomalous diffusion. Diffusion characterization is routinely performed to assess mobility in cell biology, pharmacology, and food science. Conventional FRAP is noninvasive, has low sample volume requirements, and can rapidly measure diffusion over distances of a few micrometers. However, conventional point-bleach measurements are complicated by signal-to-noise limitations, the need for precise knowledge of the bleach beam profile, potential for bias due to sample heterogeneity, and poor compatibility with multi-photon excitation due to local heating. In FT-FRAP with patterned illumination, the time-dependent fluorescence recovery signal is concentrated to puncta in the spatial Fourier domain through patterned bleaching, with substantial improvements in signal-to-noise, mathematical simplicity, representative sampling, and multiphoton compatibility. A custom nonlinear-optical beam-scanning microscope enabled patterned illumination for photobleaching through two-photon excitation. Measurements in the spatial Fourier domain removed dependence on the bleach profile, suppressing bias from imprecise knowledge of the point spread function. For normal diffusion, the fluorescence recovery produced a simple single-exponential decay in the spatial Fourier domain, in excellent agreement with theoretical predictions. Simultaneous measurement of diffusion at multiple length scales was enabled through analysis of multiple spatial harmonics of the bleaching pattern. Anomalous diffusion was characterized by FT-FRAP through a nonlinear fit to multiple spatial harmonics of the fluorescence recovery. Constraining the fit to describe diffusion over multiple length scales resulted in higher confidence in the recovered fitting parameters. Additionally, phase analysis in FT-FRAP was shown to inform on flow/sample translation. Statement of SignificanceFourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination greatly improves the accuracy of diffusion assessments and simultaneously accesses information on both normal and anomalous diffusion in a single experiment.

biophysics↗

bioRxiv2020

Microampere electric currents caused bacterial membrane damage and two-way leakage in short time

Physical agents such as low electric voltages and currents have recently gained attention for antimicrobial treatment due to their bactericidal capability. Although microampere electric currents were shown to suppress the growth of bacteria, it remains unclear to what extent the microampere currents damage bacterial membrane. Here, we investigated the membrane damage and two-way leakage caused by microampere electric currents ([≤] 100 A) in a short time (30 min). Based on MitoTracker staining, propidium iodide staining, filtration assays, and quantitative single-molecule localization microscopy, we found that microampere electric currents caused significant membrane damages and allowed two-way leakages of ions, small molecules and proteins. This study paves the way to new development and antibiotic applications of ultra-low electric voltages and currents. Statement of SignificancePrevious studies showed that treating bacteria with milliampere electric currents for 72 hours led to significant damages of the bacterial membrane. However, it remains unclear to what extent membrane damages and two-way (i.e. inward and outward) leakages are caused by lower electric currents in a shorter time. In this work, we set out to answer this question. We carried out several assays on the bacteria treated by microampere electric currents of [≤] 100 A for 30 min, including MitoTracker staining, propidium iodide staining, filtration assays, and quantitative single-molecule localization microscopy. We found and quantified that the membrane damages were caused by microampere electric currents in half an hour and allowed two-way leakages of ions, small molecules, and proteins.

biophysics↗

bioRxiv2020

Membrane Association and Functional Mechanism of Synaptotagmin-1 in Triggering Vesicle Fusion

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.

biophysics↗

bioRxiv2020

Structures of a P4-ATPase lipid flippase in lipid bilayers

Type 4 P-type ATPases (P4-ATPases) are a group of key enzymes maintaining lipid asymmetry of eukaryotic membranes. Phospholipids are actively and selectively flipped by P4-ATPases from the exoplasmic leaflet to the cytoplasmic leaflet. How lipid flipping is coupled with ATP-hydrolysis by P4-ATPases is poorly understood. Here, we report the electron cryo-microscopy structures of a P4-ATPase, Dnf1-Cdc50 from Chaetomium thermophilum, which had been reconstituted into lipid nanodiscs and captured in two transport intermediate states. The structures reveal that transmembrane segment 1 of Dnf1 becomes highly flexible during lipid transport. The local lipid bilayers are distorted to facilitate the entry of the phospholipid substrates from the exoplasmic leaflet to a cross-membrane groove. During transport, the lipid substrates are relayed through four binding sites in the groove which constantly shields the lipid polar heads away from the hydrophobic environment of the membranes.

biophysics↗

bioRxiv2020

The biomechanical role of extra-axonemal structures in shaping the flagellar beat of Euglena

We propose and discuss a model for flagellar mechanics in Euglena gracilis. We show that the peculiar non-planar shapes of its beating flagellum, dubbed "spinning lasso", arise from the mechanical interactions between two of its inner components, namely, the axoneme and the paraflagellar rod. The spontaneous shape of the axoneme and the resting shape of the paraflagellar rod are incompatible. The complex non-planar configurations of the coupled system emerge as the energetically optimal compromise between the two antagonistic components. The model is able to reproduce the experimentally observed flagellar beats and their characteristic spinning lasso geometric signature, namely, travelling waves of torsion with alternating sing along the length of the flagellum.

biophysics↗

bioRxiv2020

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

Bunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that undergoes large movements related to its role in transcription initiation and a zinc-binding domain that displays a fold not previously observed. We capture the structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational changes inherent to its function. We uncover the coordinated movement of the polymerase priming loop, lid domain and C-terminal region required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. The revealed structural details and dynamics of functional elements will be instrumental for structure-based development of compounds that inhibit RNA synthesis by the polymerase.

biophysics↗

bioRxiv2020

Impact of force function formulations on the numerical simulation of centre-based models

Centre-based, or cell-centre models are a framework for the computational study of multicellular systems with widespread use in cancer modelling and computational developmental biology. At the core of these models are the numerical method used to update cell positions and the force functions that encode the pairwise mechanical interactions of cells. For the latter there are multiple choices that could potentially affect both the biological behaviour captured, and the robustness and efficiency of simulation. For example, available open-source software implementations of centre-based models rely on different force functions for their default behaviour and it is not straightforward for a modeler to know if these are interchangeable. Our study addresses this problem and contributes to the understanding of the potential and limitations of three popular force functions from a numerical perspective. We show empirically that choosing the force parameters such that the relaxation time for two cells after cell division is consistent between different force functions results in good agreement of the population radius of a growing monolayer. Furthermore, we report that numerical stability is not sufficient to prevent unphysical cell trajectories following cell division, and consequently, that too large time steps can cause geometrical differences at the population level.

biophysics↗