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bioRxiv2018

Numerical parameter space compression and its application to microtubule dynamic instability

Physical models of biological systems can become difficult to interpret when they have a large number of parameters. But the models themselves actually depend on (i.e. are sensitive to) only a subset of those parameters. Rigorously identifying this subset of \"stiff\" parameters has been made possible by the development of parameter space compression (PSC). However, PSC has only been applied to analytically-solvable physical models. We have generalized this powerful method by developing a numerical approach to PSC that can be applied to any computational model. We validated our method against analytically-solvable models of random walk with drift and protein production and degradation. We then applied our method to an active area of biophysics research, namely to a simple computational model of microtubule dynamic instability. Such models have become increasingly complex, perhaps unnecessarily. By adding two new parameters that account for prominent structural features of microtubules, we identify one that can be \"compressed away\" (the \"seam\" in the microtubule) and another that is essential to model performance (the \"tapering\" of microtubule ends). Furthermore, we show that the microtubule model has an underlying, low-dimensional structure that explains the vast majority of our experimental data. We argue that numerical PSC can identify the low-dimensional structure of any computational model in biophysics. The low-dimensional structure of a model is easier to interpret and identifies the mechanisms and experiments that best characterize the system.

biophysics↗

bioRxiv2018

Hydrodynamic and electrophoretic properties of Trastuzumab/HER2 complexes. Experiments and modeling.

The combination of hydrodynamic and electrophoretic experiments and computer simulations is indeed a powerful approach to study the interaction between proteins. In this work, we present hydrodynamic and electrophoretic experiments in aqueous solution along with molecular dynamics and hydrodynamic modeling to monitor and compute biophysical properties of the interactions between the extracellular domain of the HER2 protein (eHER2) and the monoclonal antibody trastuzumab (TZM). The importance of this system relies on the fact that the overexpression of HER2 protein is related with the poor prognosis breast cancers (HER2++ positives) being the TZM a monoclonal antibody for the treatment of this cancer. We have found and characterize two different complexes between the TZM and eHER2 proteins (1:1 and 1:2 TZM:eHER2 complexes). The conformational features of these complexes regulate their hydrodynamic and electrostatic properties. Thus, the results indicate a high degree of molecular flexibility in the systems, that ultimately leads to higher values of the intrinsic viscosity and as well as lower values of diffusion coefficient than those expected for simple globular proteins. A highly asymmetric charge distribution is detected for the monovalent complex (1:1 complex), which has strong implications in correlations between the experimental electrophoretic mobility and the modeled net charge. In order to understand the dynamics of these systems and the role of the specific domains involved, it is essential to find biophysical correlations between dynamics, macroscopic transport and electrostatic properties. The results should be of general interest for researchers working in this area.

biophysics↗

bioRxiv2018

Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

Henipaviruses are severe human pathogens responsible for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We have previously reported a biophysical characterization of the Henipavirus V proteins and shown that they interact with DDB1, a cellular protein that is a component of the ubiquitin ligase E3 complex. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquidhydrogel phase transition. By combining experimental and bioinformatics approaches, we have identified the V region responsible for this phenomenon. This region (referred to as PNT3), which falls within the long intrinsically disordered region of V, was further investigated using a combination of biophysical and structural approaches. ThioflavinT and Congo red binding assays, together with negative-staining electron microscopy studies, show that this region forms amyloid-like, {beta}-enriched structures. Such structures are also formed in mammal cells transfected to express PNT3. Those cells also exhibit a reduced viability in the presence of a stress agent. Interestingly, mammal cells expressing a rationally designed, non-amyloidogenic PNT3 variant (PNT33A), appear to be much less sensitive to the stress agent, thus enabling the establishment of a link between fibril formation and cell toxicity. The present findings therefore pinpoint a so far never reported possible mechanism of virus-induced cell toxicity.

biophysics↗

bioRxiv2019

Cardiolipin-dependent Properties of Model Mitochondrial Membranes from Molecular Dynamics Simulations

Cardiolipin is a unique anionic lipid found in mitochondrial membranes where it contributes to various mitochondrial functions, including metabolism, mitochondrial membrane fusion/fission dynamics, and apoptosis. Dysregulation of cardiolipin synthesis and remodeling have also been implicated in several diseases, such as diabetes, heart disease and Barth Syndrome. Although cardiolipins structural and dynamic roles have been extensively studied in binary mixtures with other phospholipids, the biophysical properties of cardiolipin in ternary lipid mixtures are still not well resolved. Here, we used molecular dynamics simulations to investigate the cardiolipin-dependent properties of ternary lipid bilayer systems that mimic the major components of mitochondrial membranes. We found that changes to cardiolipin concentration only resulted in minor changes to bilayer structural features, but that the lipid diffusion was significantly affected by those alterations. We also found that cardiolipin position along the bilayer surfaces correlated to negative curvature deflections, consistent with the induction of negative curvature stress in the membrane monolayers. This work contributes to a foundational understanding of the role of CL in altering the properties in ternary lipid mixtures composed of the major mitochondrial phospholipids, providing much needed insights to help understand how cardiolipin concentration modulates the biophysical properties of mitochondrial membranes.

biophysics↗

bioRxiv2019

A game theoretic approach to deciphering the dynamics of amyloid-β aggregation along competing pathways

Aggregation of amyloid {beta} (A{beta}) peptides is a significant event that underpins Alzheimer disease (AD). A{beta} aggregates, especially the low-molecular weight oligomers, are the primary toxic agents in AD pathogenesis. Therefore, there is increasing interest in understanding their formation and behavior. In this paper, we use our previously established investigations on heterotypic interactions between A{beta} and fatty acids (FAs) that adopt off-fibril formation pathway under the control of FA concentrations, to develop a mathematical framework in defining this complex mechanism. We bring forth the use of novel game theoretic framework based on the principles of Nash equilibria to define and simulate the competing on- and off-pathways of A{beta} aggregation. Together with detailed simulations and biophysical experiments, our mathematical models define the dynamics involved in the mechanisms of A{beta} aggregation in the presence of FAs to adopt multiple pathways. Specifically, our game theoretic model indicates that the emergence of off- or on-pathway aggregates are tightly controlled by a narrow set of rate constant parameters, and one could alter such parameters to populate a particular oligomeric species. These models agree with the detailed simulations and experimental data on using FA as a heterotypic partner to modulate temporal parameters. Predicting spatiotemporal landscape along competing pathways for a given heterotypic partner such as biological lipids is a first step towards simulating physiological scenarios in which the generation of specific conformeric strains of A{beta} could be predicted. Such an approach could be profoundly significant in deciphering the biophysics of amyloid aggregation and oligomer generation, which is ubiquitously observed in many neurodegenerative diseases.

biophysics↗

bioRxiv2019

Scale-free Vertical Tracking Microscopy: Towards Bridging Scales in Biological Oceanography

Understanding key biophysical phenomena in the ocean often requires one to simultaneously focus on microscale entities, such as motile plankton and sedimenting particles, while maintaining the macroscale context of vertical transport in a highly stratified environment. This poses a conundrum: How to measure single organisms, at microscale resolution, in the lab, while allowing them to freely move hundreds of meters in the vertical direction? We present a solution in the form of a scale-free, vertical tracking microscope based on a circular \"hydrodynamic-treadmill\". Our technology allows us to transcend physiological and ecological scales, tracking organisms from marine zooplankton to single-cells over vertical scales of meters while resolving microflows and behavioral processes. We demonstrate measurements of sinking particles, including marine snow as they sediment tens of meters while capturing sub-particle-scale phenomena. We also demonstrate depth-patterned virtual-reality environments for novel behavioral analyses of microscale plankton. This technique offers a new experimental paradigm in microscale ocean biophysics by combining physiological-scale imaging with free movement in an ecological-scale patterned environment.\n\nOne sentence summaryScale-free vertical tracking microscopy captures, for the first time, untethered behavioral dynamics at cellular resolution for marine plankton.

biophysics↗

bioRxiv2019

Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins

Fluorescence fluctuation spectroscopy (FFS) can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis (SpIDA) and fluorescence moment image analysis (FMIA) are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color SpIDA (2c-SpIDA) and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering (SSF) technique to remove artifacts due to intrinsic crosstalk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species, hence this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with EGFP and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments.\n\nStatement of SignificanceFluorescence fluctuation spectroscopy (FFS) techniques determine biophysical parameters from samples containing fluorescently labeled biomolecules by considering the statistical nature of fluorescent signals measured with photodetectors. The present study introduces two-color spatial cumulant analysis (2c-SpCA) to the canon of FFS techniques. 2c-SpCA analyzes pixel-value data of two-color images collected with laser scanning fluorescence microscopes. We show that 2c-SpCA can determine several biophysical parameters in living cells including Forster resonance energy transfer efficiency, the dark state fraction of fluorescent proteins, and heteromerization between distinctly labeled proteins. In comparison to existing techniques, 2c-SpCA requires very few image frames for analysis, minimal computations, and can be applied to images of fixed tissue samples.

biophysics↗

bioRxiv2019

Effect of structure in ionized albumin - based nanoparticle: Characterisation, Emodin interaction, and in vitro cytotoxicity

A {gamma}-irradiated bovine albumin serum based nanoparticle was characterised structurally, and functionally. The nanoparticle was characterised by A.F.M, D.L.S, zeta potential, T.E.M., gel-electrophoresis, spectroscopy (UV-Vis, Fluorescence, FT-IR, and CD). Its stability was studied under adverse experimental conditions: pH values, chaotropic agents, and ionic strength and stability studies against time were mainly carried out by fluorescence spectroscopy following the changes in the tryptophan environment in the nanoparticle. Its function was studied by the interaction of the NP with the hydrophobic drug Emodin was studied. The binding and kinetic properties of the obtained complex were tested by biophysical methods as well as its toxicity in tumour cells.\n\nAccording to its biophysics, the nanoparticle is a spherical nanosized vehicle with a hydrodynamic diameter of 70 nm. Data obtained describe the nanoparticle alone as nontoxic for cancer cell lines. When combined with Emodin, the bioconjugate proved to be more active on MCF-7 and PC-3 cancer cell lines than the nanoparticle alone. No haemolytic activity was found when tested against ex vivo red blood cells. The stability of the albumin nanoparticle is based on a competition between short-range attraction forces and long-range repulsion forces. The nanoparticle showed similar behaviour as albumin against pH while improving its stability in urea and tween 80. It was stable up to 15 days and presented no protein degradation in solutions up to 2 M salt concentration. Significantly, the albumin aggregate preserves the main activity-function of albumin and improved characteristics as an excellent carrier of molecules.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC=\"FIGDIR/small/631788v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (44K):\norg.highwire.dtl.DTLVardef@154a680org.highwire.dtl.DTLVardef@7f32b2org.highwire.dtl.DTLVardef@d97fdeorg.highwire.dtl.DTLVardef@159ee36_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

The Energetics of Molecular Adaptation in Transcriptional Regulation

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.\n\nSummaryWe present a biophysical model of allosteric transcriptional regulation that directly links the location of a mutation within a repressor to the biophysical parameters that describe its behavior. We explore the phenotypic space of a repressor with mutations in either the inducer binding or DNA binding domains. Using the LacI repressor in E. coli, we make sharp, falsifiable predictions and use this framework to generate a null hypothesis for how double mutants behave given knowledge of the single mutants. Linking mutations to the parameters which govern the system allows for quantitative predictions of how the free energy of the system changes as a result, permitting coarse graining of high-dimensional data into a single-parameter description of the mutational consequences.

biophysics↗

bioRxiv2019

The development of cooperative channels explains the maturation of hair cell’s mechanotransduction

Hearing relies on the conversion of mechanical stimuli into electrical signals. In vertebrates, this process of mechano-electrical transduction (MET) is performed by specialized receptors of the inner ear, the hair cells. Each hair cell is crowned by a hair bundle, a cluster of microvilli that pivot in response to sound vibrations, causing the opening and closing of mechanosensitive ion channels. Mechanical forces are projected onto the channels by molecular springs called tip links. Each tip link is thought to connect to a small number of MET channels that gate cooperatively and operate as a single transduction unit. Pushing the hair bundle in the excitatory direction opens the channels, after which they rapidly reclose in a process called fast adaptation. It has been experimentally observed that the hair cells biophysical properties mature gradually during postnatal development: the maximal transduction current increases, sensitivity sharpens, transduction occurs at smaller hair-bundle displacements, and adaptation becomes faster. Similar observations have been reported during tip-link regeneration after acoustic damage. Moreover, when measured at intermediate developmental stages, the kinetics of fast adaptation varies in a given cell depending on the magnitude of the imposed displacement. The mechanisms underlying these seemingly disparate observations have so far remained elusive. Here, we show that these phenomena can all be explained by the progressive addition of MET channels of constant properties, which populate the hair bundle first as isolated entities, then progressively as clusters of more sensitive, cooperative MET channels. As the proposed mechanism relies on the difference in biophysical properties between isolated and clustered channels, this work highlights the importance of cooperative interactions between mechanosensitive ion channels for hearing.\n\nSIGNIFICANCEHair cells are the sensory receptors of the inner ear that convert mechanical stimuli into electrical signals transmitted to the brain. Sensitivity to mechanical stimuli and the kinetics of mechanotransduction currents change during hair-cell development. The same trend, albeit on a shorter timescale, is also observed during hair-cell recovery from acoustic trauma. Furthermore, the current kinetics in a given hair cell depends on the stimulus magnitude, and the degree of that dependence varies with development. These phenomena have so far remained unexplained. Here, we show that they can all be reproduced using a single unifying mechanism: the progressive formation of channel pairs, in which individual channels interact through the lipid bilayer and gate cooperatively.

biophysics↗

bioRxiv2019

High-yield fabrication of DNA and RNA scaffolds for single molecule force and torque spectroscopy experiments.

Single molecule biophysics experiments have enabled the observation of biomolecules with a great deal of precision in space and time, e.g. nucleic acids mechanical properties and protein-nucleic acids interactions using force and torque spectroscopy techniques. The success of these experiments strongly depends on the capacity of the researcher to design and fabricate complex nucleic acid scaffolds, as the pertinence and the yield of the experiment strongly depend on the high quality and purity of the final scaffold. Though the molecular biology techniques involved are well known, the fabrication of nucleic acids scaffold for single molecule experiments still remains a difficult task. Here, we present new protocols to generate high quality coilable double-stranded DNA and RNA, as well as DNA and RNA hairpins with ~500-1000 bp long stems. Importantly, we present a new approach based on single-stranded DNAs annealing and show, using magnetic tweezers, that it is more efficient to generate complex nucleic acid scaffolds in larger amount and at higher purity than a standard PCR-digestion-ligation approach. The protocols we describe here enable the design of any sort of complex nucleic acid scaffold for single molecule biophysics experiments and will therefore be extremely valuable to the community.

biophysics↗

bioRxiv2019

Kinetic mechanisms of fast glutamate sensing by fluorescent protein probes

Protein-based fluorescent glutamate sensors have the potential for real-time monitoring of synaptic and cellular glutamate concentration changes, however even the fastest currently available sensors response times of 2-3 ms are too slow for accurate reporting of the post-synaptic AMPA receptor function in physiological conditions. We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding: affinity variants of iGluSnFR termed iGluh, iGlum and iGlul covering a range of Kd-s (5.8 M, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP with a Kd of 9.7 M are presented. The fluorescence response kinetics of all the probes are consistent with two-step mechanisms involving ligand binding and rate limiting isomerisation, however the contribution in each step to the total fluorescence enhancement and kinetic paths to the final state are diverse. In contrast to the previously characterised ultrafast indicators iGluu and iGluf, for which fluorescence enhancement occurred only in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with a significant fraction of the fluorescence increase evoked by glutamate binding which, in the case of iGluh and Fl-GluBP, occurs with a diffusion limited rate constant. The above genetically encoded and chemically labelled fluorescent glutamate sensor variants demonstrate how single amino acid changes around the binding site introduce structural heterogeneity affecting the kinetic mechanism of interactions with glutamate. Through their broad affinity range and mechanistic variety, the probes contribute to a novel toolkit for monitoring processes of glutamate neurotransmission and cellular homeostasis.\n\nSTATEMENT OF SIGNIFICANCEGlutamate is a major excitatory neurotransmitter, important in synaptic plasticity e.g. memory formation. Although predicted to clear rapidly from the synaptic cleft following presynaptic release, optical monitoring of glutamate neurotransmission has only become possible with the advent of fluorescent, protein-based indicators. Understanding their biophysical properties is important for quantification of the observed processes. Here we report the biophysical characterisation of a number of glutamate indicator variants based on the bacterial periplasmic glutamate/aspartate binding protein, revealing the subtle differences in their kinetic pathway caused by structural alteration of the glutamate binding protein by point mutations. Diffusion limited glutamate binding indicated by a novel chemically labelled probe hints at the mechanism that underlies the rapid response of the AMPA receptor.

biophysics↗

bioRxiv2019

Variable Mutations at the p53-R273 Oncogeic Hotspot Position Leads to Altered Properties

Mutations in p53 protein, especially in the DNA binding domain is one of the major hallmarks of cancer. The R273 position is a DNA contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant-variants of R273 in p53 have different survival rates indicating that the DNA contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the structural properties of three major oncogenic variants of the R273: ([R273L], [R273H], and [R273C])p53. Using a series of biophysical, biochemical and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but also show distinct structural stabilty, aggregation and toxicity profiles. [R273C]p53 shows maximum amyloidogenicity while [R273L]p53 shows maximum aggregation. Further probe in the aggregation mechanism show that [R273C]p53 aggregation is disulphide mediated whereas hydrophobic interactions dominate self-assembly in [R273L]p53. MD simulation studies clearly show that -helical intermediates are observed in [R273C]p53 whereas {beta}-sheets are observed for [R273L]p53. Our study indicates that each of the R273 variant has its own distinct property of stability and self-assembly, the molecular basis of which, may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue specific or process specific protein aggregation as target.\n\nStatement of significanceThe present work stems from an interesting observation that genetic mutations that results in switching of one amino acid to different variants at the same codon show different cancer cell progression. We are trying to understand the molecular reason behind the different gain-of-function opted by these variants. With the help of biophysical and biochemical experiments, and computational studies we have observed that the different thermal stability, unique mechanism of unfolding and self-assembly might be one of the crucial parameters for their different oncogenic effect. These studies thus call for the need of developing therapeutic strategies that consider the resultant mutant-variant as a target rather than mutation position. This is an important lead towards the understanding of cancer.

biophysics↗

bioRxiv2019

Structure-based screening of binding affinities via small-angle X-ray scattering

Protein-protein and protein-ligand interactions can alter the scattering properties of participating molecules, and thus be quantified by solution small-angle X-ray scattering (SAXS). In such cases, scattering reveals structural details of the bound complex, number of species involved, and in principle strength of the interaction. However, determining binding affinities from SAXS-based titrations is not yet an established procedure with well-defined performance expectations. We thus used periplasmic binding proteins and in particular histidine-binding protein as a standard reference, then examined precision and accuracy of affinity prediction at multiple concentrations and exposure times. By analyzing several structural and comparative scattering metrics, we found that the volatility of ratio between titrated scattering curves and a common reference most reliably quantifies ligand-triggered changes. This ratio permits the determination of affinities at low signal-to-noise ratios and without pre-determining the complex scattering, demonstrating that SAXS-based ligand screening is a promising alternative biophysical method for drug discovery pipelines.\n\nSIGNIFICANCESolution X-ray scattering can be used to screen a set of biomolecular interactions, which yields quantitative information on both structural changes and dissociation constants between binding partners. However, no common benchmarks yet exist for the application of SAXS within drug discovery workflows. Thus, investigations into its performance limitations are currently needed to make SAXS a reliable source for high-throughput screening. This study establishes a generalizable protocol based on protein-ligand interactions, and demonstrates its reproducibility across several beamline setups. In the simplest case, the micromolar binding affinities can be determined directly from measured intensities without knowledge of the molecular structure, with material consumption that is competitive with other biophysical screening techniques.

biophysics↗

bioRxiv2019

Stress generation, relaxation and size control in confined tumor growth

Experiments on tumor spheroids have shown that compressive stress from their environment can reversibly decrease tumor expansion rates and final sizes. Stress release experiments show that nonuniform anisotropic elastic stresses can be distributed throughout. The elastic stresses are maintained by structural proteins and adhesive molecules, and can be actively relaxed by a variety of biophysical processes. In this paper, we present a new continuum model to investigate how the growth-induced elastic stresses and active stress relaxation, in conjunction with cell size control feedback machinery, regulate the cell density and stress distributions within growing tumors as well as the tumor sizes in the presence of external physical confinement and gradients of growth-promoting chemical fields. We introduce an adaptive reference map that relates the current position with the reference position but adapts to the current position in the Eulerian frame (lab coordinates) via relaxation. This type of stress relaxation is similar to but simpler than the classical Maxwell model of viscoelasticity in its formulation. By fitting the model to experimental data from two independent studies of tumor spheroid growth and their cell density distributions, treating the tumors as incompressible, neo-Hookean elastic materials, we find that the rates of stress relaxation of tumor tissues can be comparable to volumetric growth rates. Our study provides insight on how the biophysical properties of the tumor and host microenvironment, mechanical feedback control and diffusion-limited differential growth act in concert to regulate spatial patterns of stress and growth. When the tumor is stiffer than the host, our model predicts tumors are more able to change their size and mechanical state autonomously, which may help to explain why increased tumor stiffness is an established hallmark of malignant tumors. Author summaryThe mechanical state of cells can modulate their growth and division dynamics via mechanotransduction, which affects both the cell size distribution and the tissue size as a whole. Experiments on tumor spheroids have shown that compressive stress from their environment can reversibly decrease tumor expansion rates and final sizes. Besides external confinement and compression on the tumor border, a heterogeneous stress field can be generated inside the tumor by nutrient-driven differential growth. Such growth-induced mechanical stresses can be relaxed by tissue rearrangement, which happens during cell neighbor exchanges, cell divisions, and extracellular matrix renewal. In this study, we have developed a continuum model that describes the above mechanical interactions and the dynamics of tissue rearrangement explicitly. Motivated by published experimental data, we consider mechanotransduction where the local compressive stress slows down cell growth and cell size reduction limits cell division. We have analyzed how external mechanical stimuli and internal processes influence the outcome of cell-and-tissue sizes and spatial patterns of cell density and mechanical stress in growing tumors.

biophysics↗

bioRxiv2019

Nonequilibrium Theory of Epigenomic Microphase Separation in the Cell Nucleus

Understanding the spatial organisation of the genome in the cell nucleus is one of the current grand challenges in biophysics. Certain biochemical - or epigenetic - marks that are deposited along the genome are thought to play an important, yet poorly understood, role in determining genome organisation and cell identity. The physical principles underlying the interplay between epigenetic dynamics and genome folding remain elusive. Here we propose and study a theory that assumes a coupling between epigenetic mark and genome densities, and which can be applied at the scale of the whole nucleus. We show that equilibrium models are not compatible with experiments and a qualitative agreement is recovered by accounting for non-equilibrium processes which can stabilise microphase separated epigenomic domains. We finally discuss the potential biophysical origin of these terms.

biophysics↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗

bioRxiv2019

Sequence-structure-function relationships in class I MHC: a local frustration perspective

Class I Major Histocompatibility Complex (MHC) binds short antigenic peptides with the help of Peptide Loading Complex (PLC), and presents them to T-cell Receptors (TCRs) of cytotoxic T-cells and Killer-cell Immunglobulin-like Receptors (KIRs) of Natural Killer (NK) cells. With more than 10000 alleles, the Human Leukocyte Antigen (HLA) chain of MHC is the most polymorphic protein in humans. This allelic diversity provides a wide coverage of peptide sequence space, yet does not affect the three-dimensional structure of the complex. Moreover, TCRs mostly interact with pMHC in a common diagonal binding mode, and KIR-pMHC interaction is allele-dependent. With the aim of establishing a framework for understanding the relationships between polymorphism (sequence), structure (conserved fold) and function (protein interactions) of the MHC, we performed here a local frustration analysis on pMHC homology models covering 1436 HLA I alleles. An analysis of local frustration profiles indicated that (1) variations in MHC fold are unlikely due to minimally-frustrated and relatively conserved residues within the HLA peptide-binding groove, (2) high frustration patches on HLA helices are either involved in or near interaction sites of MHC with the TCR, KIR, or Tapasin of the PLC, and (3) peptide ligands mainly stabilize the F-pocket of HLA binding groove. Author SummaryA protein complex called the Major Histocompatibility Complex (MHC) plays a critical role in our fight against pathogens via presentation of antigenic peptides to receptor molecules of our immune system cells. Our knowledge on genetics, structure and protein interactions of MHC revealed that the peptide-binding groove of Human Leukocyte Chain (HLA I) of this complex is highly polymorphic and interacts with different proteins for peptide-binding and presentation over the course of its lifetime. Although the relationship between polymorphism and peptide-binding is well-known, we still lack a proper framework to understand how this polymorphism affects the overall MHC structure and protein interactions. Here, we used computational biophysics methods to generate structural models of 1436 HLA I alleles, and quantified local frustration within the HLA I, which indicates energetic optimization levels of contacts between amino acids. We identified a group of minimally frustrated and conserved positions which may be responsible for the conserved MHC structure, and detected high frustration patches on HLA surface positions taking part in interactions with other immune system proteins. Our results provide a biophysical basis for relationships between sequence, structure, and function of MHC I.

biophysics↗