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bioRxiv2017

Epigenetic Transitions And Knotted Solitons In Stretched Chromatin

The spreading and regulation of epigenetic marks on chromosomes is crucial to establish and maintain cellular identity. Nonetheless, the dynamical mechanism leading to the establishment and maintenance of a given, cell-line specific, epigenetic pattern is still poorly understood. In this work we propose, and investigate in silico, a possible experimental strategy to illuminate the interplay between 3D chromatin structure and epigenetic dynamics. We consider a set-up where a reconstituted chromatin fibre is stretched at its two ends (e.g., by laser tweezers), while epigenetic enzymes (writers) and chromatin-binding proteins (readers) are flooded into the system. We show that, by tuning the stretching force and the binding affinity of the readers for chromatin, the fibre undergoes a sharp transition between a stretched, epigenetically disordered, state and a crumpled, epigenetically coherent, one. We further investigate the case in which a knot is tied along the chromatin fibre, and find that the knotted segment enhances local epigenetic order, giving rise to \"epigenetic solitons\" which travel and diffuse along chromatin. Our results point to an intriguing coupling between 3D chromatin topology and epigenetic dynamics, which may be investigated via single molecule experiments.

biophysics↗

bioRxiv2017

Torque and buckling in stretched intertwined double-helix DNAs

We present a statistical-mechanical model for the behavior of intertwined DNAs, with a focus on their torque and extension as a function of their catenation (linking) number and applied force, as studied in magnetic tweezers experiments. Our model produces results in good agreement with available experimental data, and predicts a catenation-dependent effective twist modulus distinct from what is observed for twisted individual double-helix DNAs. We find that buckling occurs near to the point where experiments have observed a kink in the extension versus linking number, and that the subsequent \"supercoiled braid\" state corresponds to a proliferation of multiple small plectoneme structures. We predict a discontinuity in extension at the buckling transition corresponding to nucleation of the first plectoneme domain. We also find that buckling occurs for lower linking number at lower salt; the opposite trend is observed for supercoiled single DNAs.

biophysics↗

bioRxiv2017

Millisecond Dynamics Of BTK Reveal Kinome-Wide Conformational Plasticity Within The Apo Kinase Domain

Bruton tyrosine kinase (BTK) is a key enzyme in B-cell development whose improper regulation causes severe immunodeficiency diseases. Design of selective BTK therapeutics would benefit from improved, in-silico structural modeling of the kinases solution ensemble. However, this remains challenging due to the immense computational cost of sampling events on biological timescales. In this work, we combine multi-millisecond molecular dynamics (MD) simulations with Markov state models (MSMs) to report on the thermodynamics, kinetics, and accessible states of BTKs kinase domain. Our conformational landscape links the active state to several inactive states, connected via a structurally diverse intermediate. Our calculations predict a kinome-wide conformational plasticity, and indicate the presence of several new potentially druggable BTK states. We further find that the population of these states and the kinetics of their inter-conversion are modulated by protonation of an aspartate residue, establishing the power of MD & MSMs in predicting effects of chemical perturbations.

biophysics↗

bioRxiv2017

Facilitated Dissociation Of Transcription Factors From Single DNA Binding Sites

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate {bsim}1 x 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.\n\nSIGNIFICANCE STATEMENTTranscription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.\n\nAuthor ContributionsRamsey I. Kamar\n\ndesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper\n\nEdward J. Banigan\n\ndesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper\n\nAykut Erbas\n\ndesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper\n\nRebecca D. Giuntoli\n\ndesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper\n\nMonica Olvera de la Cruz\n\ndesigned research, performed research, wrote the paper\n\nReid C. Johnson\n\ndesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper\n\nJohn F. Marko\n\ndesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

biophysics↗

bioRxiv2017

Pushing The Limits Of Detection Of Weak Binding Using Fragment Based Drug Discovery: Identification Of New Cyclophilin Binders

Fragment Based Drug Discovery (FBDD) is an increasingly popular method to identify novel small-molecule drug candidates. One of the limitations of the approach is the difficulty of accurately characterizing weak binding events. This work reports a combination of X-ray diffraction, surface plasmon resonance (SPR) experiments and molecular dynamics (MD) simulations, for the characterisation of binders to different isoforms of the cyclophilin (Cyp) protein family. Although several Cyp inhibitors have been reported in the literature, it has proven challenging to achieve high binding selectivity for different isoforms of this protein family. The present studies have led to the identification of several structurally novel fragments that bind to diverse Cyp isoforms in distinct pockets with low millimolar dissociation constants. A detailed comparison of the merits and drawbacks of the experimental and computational techniques is presented, and emerging strategies for designing ligands with enhanced isoform specificity are described.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC=\"FIGDIR/small/136101_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@5d2d48org.highwire.dtl.DTLVardef@147f922org.highwire.dtl.DTLVardef@356562org.highwire.dtl.DTLVardef@1933853_HPS_FORMAT_FIGEXP M_FIG C_FIG Research Highlights O_LIFBDD is a popular method but weak binding is difficult to detect\nC_LIO_LIThere is a need for pushing the limits of weak binding detection\nC_LIO_LICombination of X-ray, SPR and MD methodologies increases successful characterization of weak binding events\nC_LIO_LISeveral novel Cyclophilin fragment binders were identified\nC_LI

biophysics↗

bioRxiv2017

A Systematic Analysis Of Atomic Protein-Ligand Interactions In The PDB

As the protein databank (PDB) recently passed the cap of 123,456 structures, it stands more than ever as an important resource not only to analyze structural features of specific biological systems, but also to study the prevalence of structural patterns observed in a large body of unrelated structures, that may reflect rules governing protein folding or molecular recognition. Here, we compiled a list of 11,016 unique structures of small-molecule ligands bound to proteins - 6,444 of which have experimental binding affinity - representing 750,873 protein-ligand atomic interactions, and analyzed the frequency, geometry and impact of each interaction type. We find that hydrophobic interactions are generally enriched in high-efficiency ligands, but polar interactions are over-represented in fragment inhibitors. While most observations extracted from the PDB will be familiar to seasoned medicinal chemists, less expected findings, such as the high number of C-H ...O hydrogen bonds or the relatively frequent amide-{pi} stacking between the backbone amide of proteins and aromatic rings of ligands, uncover underused ligand design strategies.

biophysics↗

bioRxiv2017

Element-Free Multiscale Modeling Of Large Deformation Behavior Of Red Blood Cell Membrane With Malaria Infection

In normal physiological and healthy conditions, red blood cells (RBCs) deform readily as they pass through the microcapillaries and the spleen. In this paper, we examine the effects of Plasmodium falciparum infection and maturation on the large deformation behavior of malaria-infected red blood cells (iRBCs) by means of a three-dimensional (3D) multiscale meshfree method. We numerically simulated the optical tweezers experiment and observed the force-displacement response of the iRBC membrane as malaria infection progresses. Our simulation results agree well with experimental data and confirm that the deformability of malaria-infected cells decreases significantly as malaria infection progresses.

biophysics↗

bioRxiv2017

Boosting The Localization Precision Of dSTORM By Biocompatible Metal-Dielectric Coated Glass Coverslips

Super-resolution techniques such as direct Stochastic Optical Reconstruction Microscopy (dSTORM) have become versatile and well-established tools for biological imaging over the last century. Here, we theoretically and experimentally show that clever combination of different fluorescence modalities allows further improvements. We found that the interaction of fluorophores with plasmonic surfaces boost super-resolution performance in dSTORM approaches as it allows for tailoring the excitation and emission properties. The strength of the approach is that no further specialized microscope setup is required as the described enhancement solely rely on metal-dielectric coated glass coverslips that are straightforward to fabricate. Such biocompatible plasmonic nanolayers enhance the signal-to-noise ratio of dSTORM, and thus sharpens the localization precision by a factor of two.

biophysics↗

bioRxiv2017

Does Temperature Change Worsen Or Mitigate The Effect Of Malaria Infection On Erythrocyte Deformability?

INTRODUCTION INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES This paper attempts to investigate whether temperature change worsen or mitigate the effect of Plasmodium falciparum (P. falciparum) infection and maturation on the deformability of malaria-infected red blood cells (iRBCs) or erythrocytes. P. falciparum infection is the most lethal malaria parasitic infection affecting humans [1]. Upon infection, the merozoites enter the bloodstream and develop through the ring (Pf-rRBC), trophozoite (Pf-tRBC) and schizont (Pf-tRBC) stages, which is usually accompanied by significant changes in body temperature. Hence, this study aims to draw a connection between temperature change and erythrocyte deformability upon malaria infection. ...

biophysics↗

bioRxiv2017

Response-Time Behaviors Of Intercellular Communication Network Motifs

Cell-to-cell communication networks have critical roles in diverse organismal processes, such as coordinating tissue development or immune cell response. However, compared to intracellular signal transduction networks, the function and engineering principles of cell-to-cell communication networks are far less understood. Here, we study cell-to-cell communication networks using a framework that models the input-to-output relationship of intracellular signal transduction networks with a single function--the response-time distribution. We identify a prototypic response-time distribution--the gamma distribution--arising in both experimental data sets and mathematical models of signal-transduction pathways. We find that simple cell-to-cell communication circuits can generate bimodal response-time distributions, and can control synchronization and delay of cell-population responses independently. We apply our modeling approach to explain otherwise puzzling data on cytokine secretion onset times in T cells. Our approach can be used to predict communication network structure using experimentally accessible input-to-output measurements and without detailed knowledge of intermediate steps.

biophysics↗

bioRxiv2017

Gaussian Curvature Directs The Distribution Of Spontaneous Curvature On Bilayer Membrane Necks

Formation of membrane necks is crucial for fission and fusion in lipid bilayers. In this work, we seek to answer the following fundamental question: what is the relationship between protein-induced spontaneous mean curvature and the Gaussian curvature at a membrane neck? Using an augmented Helfrich model for lipid bilayers to include membrane-protein interaction, we solve the shape equation on catenoids to find the field of spontaneous curvature that satisfies mechanical equilibrium of membrane necks. In this case, the shape equation reduces to a variable coefficient Helmholtz equation for spontaneous curvature, where the source term is proportional to the Gaussian curvature. We show how this latter quantity is responsible for non-uniform distribution of spontaneous curvature in minimal surfaces. We then explore the energetics of catenoids with different spontaneous curvature boundary conditions and geometric asymmetries to show how heterogeneities in spontaneous curvature distribution can couple with Gaussian curvature to result in membrane necks of different geometries.

biophysics↗

bioRxiv2017

The Condensin Complex Is A Mechanochemical Motor That Translocates Along DNA

One Sentence SummarySingle-molecule imaging reveals that eukaryotic condensin is a highly processive DNA-translocating motor complex.\n\nAbstractCondensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. Here, we use single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Condensins translocation activity is rapid and highly processive, with individual complexes traveling an average distance of [≥]10 kilobases at a velocity of [~]60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its [~]50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.

biophysics↗

bioRxiv2017

Conformational Dynamics In TRPV1 Channels Reported By An Encoded Coumarin Amino Acid

Transient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a bundle crossing at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

biophysics↗

bioRxiv2017

Defocus and magnification dependent variation of TEM image astigmatism

Daily alignment of the microscope is a prerequisite to reaching optimal illumination lens and imaging lens conditions for high resolution imaging in cryo-EM. In contrast to the dramatic progress in automated image acquisition and post-image processing techniques, less attention has been paid to the improvement of microscope alignment before data collection. In this study, we have employed our recently published tool, s2stigmator, to study how image astigmatism varies with the imaging conditions (e.g. defocus, magnification). We have found that the large change of defocus/magnification between visual correction of astigmatism and subsequent data collection tasks, or even during data collection will inevitably result in undesirable residual astigmatism in the final images. Furthermore, the dependence of astigmatism on the imaging conditions varies significantly from time to time, so that it cannot be reliably compensated by pre-calibration of the microscope. These findings have essentially invalidated a basic assumption of current cryo-EM imaging strategies that assumes invariant astigmatism for different defocuses/magnifications used in the microscope alignment stage and the final data acquisition stage. Based on these findings, we recommend the same magnification and the median defocus of the intended defocus range for final data collection are used in the objective lens astigmatism correction task during microscope alignment and in the focus mode of the iterative low-dose imaging. It is also desirable to develop a fast, accurate method that can perform dynamic correction of the astigmatism for different intended defocuses during automated imaging.

biophysics↗

bioRxiv2017

The AWSEM-Amylometer: Predicting Amyloid Propensity And Fibril Topology Using An Optimized Folding Landscape Model

Amyloids are fibrillar protein aggregates with simple repeated structural motifs in their cores, usually {beta}-strands but sometimes -helices. Identifying the amyloid-prone regions within protein sequences is important both for understanding the mechanisms of amyloid-associated diseases and for understanding functional amyloids. Based on the crystal structures of seven cross-{beta} amyloidogenic peptides with different topologies and one recently solved cross- fiber structure, we have developed a computational approach for identifying amyloidogenic segments in protein sequences using the Associative memory, Water mediated, Structure and Energy Model. The AWSEM-Amylometer performs favorably in comparison with other predictors in predicting aggregation-prone sequences in multiple datasets. The method also predicts the specific topologies (the relative arrangement of {beta}-strands in the core) of the amyloid fibrils well. An important advantage of the AWSEM-Amylometer over other existing methods is its direct connection with an efficient, optimized protein folding simulation model, AWSEM. This connection allows one to combine efficient and accurate search of protein sequences for amyloidogenic segments with the detailed study of the thermodynamic and kinetic roles that these segments play in folding and aggregation in the context of the entire protein sequence. We present new simulation results that highlight the free energy landscapes of peptides that can take on multiple fibril topologies. We also demonstrate how the Amylometer methodology can be straightforwardly extended to the study of functional amyloids that have the recently discovered cross- fibril architecture.

biophysics↗

bioRxiv2017

Poor man's two photon imaging: Scanning Laser Upconversion Microscopy

Two photon microscopy is one of the most powerful techniques for optical images. Its deep penetration, low scattering and mainly its sectioning power allows exquisite control on all three axes of 3D samples, both for imaging and actuation. Unfortunately, this technique involves very expensive femtosecond lasers. Upconverting Nanoparticles display multiphoton absorption at low instantaneous power, which can be achieved by means of cheap laser diodes. Scanning Laser Upconversion Microscopy offers the deep penetration and low scattering of NIR excitation together with the sectioning power of multiphoton techniques for under US$ 1000.

biophysics↗

bioRxiv2017

A Liquid Chromatography-Mass Spectrometry Method For Screening Disulfide Tethering Fragments

We report the refinement of a high-throughput, liquid-chromatography/mass spectrometry (LC/MS)-based screening method for the identification of covalent small-molecule binders to proteins. Using a custom library of 1600 disulfide-capped fragments targeting surface cysteine residues, we optimize sample preparation, chromatography, and ionization conditions to maximize the reliability and flexibility of the approach. Data collection at a rate of 90 seconds per sample balances speed and reliability for sustained screening over multiple, diverse projects run over a 24-month period. The method is applicable to protein targets of various classes and a range of molecular masses. Data are processed in a custom pipeline that calculates a % bound value for each compound and detects false-positives by calculating significance of detected masses ( signal significance). An example pipeline has been made available through Biovias ScienceCloud Protocol Exchange. Data collection and analysis methods for the screening of covalent adducts of intact proteins are now fast enough to screen the largest covalent compound libraries in 1-2 days.

biophysics↗

bioRxiv2017

A Combined Use Of Intravoxel Incoherent Motion MRI Parameters Can Differentiate Early Stage Hepatitis-B Fibrotic Livers From Healthy Livers

This study investigated a combined use of IVIM parameters Dslow (D), PF (f) and Dfast (D*) for liver fibrosis evaluation. 16 healthy volunteers (F0) and 33 hepatitis-b patients (stage F1= 15, stage F2-4 = 18) were included. With a 1.5-T MR scanner and respiration-gating, IVIM diffusion weighted imaging was acquired using a single-shot echo-planar imaging sequence with ten b-values of 10, 20, 40, 60, 80, 100, 150, 200, 400, and 800 s/mm2. Signal measurement was performed on right liver parenchyma. With a 3-dimensional tool, Dslow, PF, and Dfast values were placed along the x-axis, y-axis, and z-axis, and a plane was defined to separate healthy volunteers from patients. 3-dimensional tool demonstrated healthy volunteers and all patients with liver fibrosis could be separated. Classification and Regression Tree showed a combination of PF (PF < 12.55%), Dslow (Dslow < 1.152 x10-3 mm2/s) and Dfast (Dfast <13.36 x10-3 mm2/s) could differentiate healthy subjects and all fibrotic livers (F1-F4) with an area under the curve of logistic regression (AUC) of 0.986. The AUC for differentiation of healthy livers vs. F2-4 livers was 1. PF offered the best diagnostic value, followed by Dslow; however, all three parameters of PF, Dslow, and Dfast contributed to liver fibrosis detection.

biophysics↗