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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

The Effects Of Oncogenic G12D Mutation On K-Ras Structure, Conformation And Dynamics

K-Ras is the most frequently mutated oncoprotein in human cancers, and G12D is its most prevalent mutation. To understand how G12D mutation impacts K-Ras function, we need to understand how it alters the regulation of its dynamics. Here, we present local changes in K-Ras structure, conformation and dynamics upon G12D mutation, from long-timescale Molecular Dynamics simulations of active (GTP-bound) and inactive (GDP-bound) forms of wild-type and mutant K-Ras, with an integrated investigation of atomistic-level changes, local conformational shifts and correlated residue motions. Our results reveal that the local changes in K-Ras are specific to bound nucleotide (GTP or GDP), and we provide a structural basis for this. Specifically, we show that G12D mutation causes a shift in the population of local conformational states of K-Ras, especially in Switch-II (SII) and 3-helix regions, in favor of a conformation that is associated with a catalytically impaired state through structural changes; it also causes SII motions to anti-correlate with other regions. This detailed picture of G12D mutation effects on the local dynamic characteristics of both active and inactive protein helps enhance our understanding of local K-Ras dynamics, and can inform studies on the development of direct inhibitors towards the treatment of K-RasG12D-driven cancers.

biophysics↗

bioRxiv2017

PDB-NMA of a Protein Homodimer Reproduces Distinct Experimental Motility Asymmetry

We have extended our analytically derived PDB-NMA formulation, ATMAN [1], to include protein dimers using mixed internal and Cartesian coordinates. A test case on a 1.3[A] resolution model of a small homodimer, ActVA-ORF6, consisting of two 112-residue subunits identically folded in a compact 50[A] sphere, reproduces the distinct experimental Debye-Waller motility asymmetry for the two chains, demonstrating that structure sensitively selects vibrational signatures. The vibrational analysis of this PDB entry, together with biochemical and crystallographic data, demonstrates the cooperative nature of the dimeric interaction of the two subunits and suggests a mechanical model for subunit interconversion during the catalytic cycle.

biophysics↗

bioRxiv2017

Microscopic Dissipative Structuring at the Origin of Life

Fundamental molecules of life are suggested to be formed, proliferated, and evolved through microscopic dissipative structuring and autocatalytic replication under the UV-C solar spectrum prevalent at Earths surface throughout the Archean. Evidence is given in the numerous salient characteristics of these, including their strong absorption in this spectral region, their rapid non-radiative decay through an inherent conical intersection, UV-C activation (phos-phorylation) of nucleotides, and UV-C induced denaturing of double helix RNA and DNA. The examples of the dissipative structuring and dissipative proliferation of the purines and of single strand DNA are given. This provides a physical-chemical foundation for understanding the origin and evolution of life.

biophysics↗

bioRxiv2017

IVIM parameters have good scan-rescan reproducibility when evidential motion contaminated and poorly fitted image data are removed

BackgroundIntravoxel Incoherent Motion (IVIM) diffusion MRI is a promising technique for liver pathology evaluation, but this techniques scan-rescan reproducibility has been reported to be unsatisfactory.\n\nObjectiveTo understand whether IVIM MRI parameters for liver parenchyma can be good after removal of motion contaminated and/or poorly fitted image data.\n\nMaterial and MethodsEighteen healthy volunteers had liver scanned twice at the same session to assess scan-rescan repeatability, and again in another session after an average interval of 13 days to assess reproducibility. Diffusion weighted image were acquired with a 3T scanner using respiratory-triggered echo-planar sequence and 16 b-values (0 to 800 s/mm2). Measurement was performed on the right liver with segmented-unconstrained least square fitting. Image series with evidential anatomical mismatch, apparent artifacts, and poorly fitted signal intensity vs. b-value curve were excluded. A minimum of three slices was deemed necessary for IVIM parameter estimation of a liver.\n\nResultsWith total 54 examinations, 6 scans did not satisfy inclusion criteria, leading to a success rate of 89%; and 14 volunteers were finally included. With each scan a mean of 5.3 slices (range: 3-10 slices) were utilized for analysis. Using threshold b-value=80s/mm2, the coefficient of variation and within-subject coefficient of variation for repeatability and reproducibility were: 2.86% and 4.24% for Dslow, 3.81% and 4.24%, for PF, 18.16% and 24.88% for Dfast; and those for reproducibility were 2.48% and 3.24% for Dslow; 4.91% and 5.38% for PF; 21.18% and 30.89% for Dfast.\n\nConclusionIVIM parameter scan-rescan reproducibility can be potentially good.

biophysics↗

bioRxiv2017

Ultra-fast super-resolution imaging of biomolecular mobility in tissues

Super-resolution techniques have addressed many biological questions, yet molecular quantification at rapid timescales in live tissues remains challenging. We developed a light microscopy system capable of sub-millisecond sampling to characterize molecular diffusion in heterogeneous aqueous environments comparable to interstitial regions between cells in tissues. We demonstrate our technique with super-resolution tracking of fluorescently labelled chemokine molecules in a collagen matrix and ex vivo lymph node tissue sections, outperforming competing methods.

biophysics↗

bioRxiv2017

DNA sequence encodes the position of DNA supercoils

The three-dimensional structure of DNA is increasingly understood to play a decisive role in gene regulation and other vital cellular processes, which has triggered an explosive growth of research on the spatial architecture of the genome. Many studies focus on the role of various DNA-packaging proteins, crowding, and confinement in organizing chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we use a fluorescence-based single-molecule technique to visualize plectonemes, the extended intertwined DNA loops that form upon twisting DNA. Remarkably, we find that the underlying DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific positions. We explore a variety of DNA sequences to determine what features influence pinning, and we develop a physical model that predicts the level of plectoneme pinning in excellent agreement with the data. The intrinsic curvature measured over a range of ~70 base pairs is found to be the key property governing the supercoiled structure of DNA. Our model predicts that plectonemes are likely to localize directly upstream of prokaryotic transcription start sites, and this prediction is experimentally verified in vitro. Our results reveal a hidden code in DNA that helps to spatially organize the genome.

biophysics↗

bioRxiv2017

Motor reattachment kinetics play a dominant role in multimotor-driven cargo transport

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multi-motor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be 4-fold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of gold-nanoparticle-labeled cargo with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than do kinesin-1. Finally, cargo transported by kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multi-motor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads while kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

biophysics↗

bioRxiv2017

Seamless Multiscale Modeling of Coagulation Using Dissipative Particle Dynamics

We propose a new multiscale framework that seamlessly integrates four key components of blood clotting namely, blood rheology, cell mechanics, coagulation kinetics and transport of species and platelet adhesive dynamics. We use transport dissipative particle dynamics (tDPD), which is the extended form of original DPD, as the base solver, while a coarse-grained representation of blood cells membrane accounts for its mechanics. Our results show the dominant effect of blood flow and high Peclet numbers on the reactive transport of the chemical species signifying the importance of membrane bound reactions on the surface of adhered platelets. This new multiscale particle-based methodology helps us probe synergistic mechanisms of thrombus formation, and can open new directions in addressing other biological processes from sub-cellular to macroscopic scales.

biophysics↗

bioRxiv2017

A "Tug of War" maintains a dynamic protein-membrane complex: MD simulations of C-Raf RBD-CRD bound to K-Ras4B at an anionic membrane

Association of Raf kinase with activated Ras triggers downstream signaling cascades, towards regulating transcription in the cells nucleus. Dysregulation of Ras: Raf signaling stimulates cancers. We investigate the C-Raf RBD and CRD regions when bound to oncogenic K-Ras4B at the membrane. All-atom molecular dynamics simulations suggest that the membrane plays an integral role in regulating the configurational ensemble of the complex. Remarkably, the complex samples a few states dynamically, reflecting a competition between C-Raf CRD and K-Ras4B- membrane interactions. This competition arises because the interaction between the RBD and K-Ras is strong and the linker between the RBD and CRD is short. This study reveals a mechanism that maintains a modest binding for the overall complex at the membrane to facilitate fast signaling processes. It is likely a common mechanism for other multi-protein, if not multidomain proteins at membranes.

biophysics↗

bioRxiv2017

‘Switch-like’ transition from random to directed motility of microtubules by a yeast dynein

Processive transport by multiple molecular motors that step stochastically, requires a form of mechanical coupling. In a quantitative microtubule (MT) gliding assay with yeast cytoplasmic dynein, we investigate the nature of this coupling by examining the effect of MT length and motor density on transport. We find speed and velocity have a length dependence for low motor numbers, but are independent of MT length for high motor densities. The dependence of speed, velocity and degree of randomness of MT transport is best understood when evaluated in terms of the numbers of motors bound to a filament. A model of collective transport of MTs, based on stochastic stepping and asymmetric detachment rates, reproduces the experimental trends of decreasing diffusivity with increasing number of motors. Additionally, the model predicts a switch-like increase in directionality of MT transport above a threshold number of motors. Such a rapid transition from random to directed motility with increasing numbers of yeast dyneins, could play a role in vivo during mitosis in the search and orientation of the Saccharomyces cerevisiae nucleus.

biophysics↗

bioRxiv2017

Optical Coherence Tomography Reveals Mechanobiologically Stable Self-Organizing Di-Fork Architecture of Mice Cutaneous Scars

Scientific studies report crucial impacts of biomechanical effectors to modulate wound healing either by scarring or regeneration. Further, the biological decision to predominantly favor the former is still cryptic. Real-time visualization of biomechanical manifestations in situ in scarring is hence necessary. Endorsed by nanostructural testing, synthetic phantom analysis, and computational simulations, we found strong mechanobiological correlates for Swept Source Optical Coherence Tomography (SS-OCT) speckles in mice cutaneous repair (full-thickness) up to 10 months. The theoretical basis of the optomechanics to provide insights into scar form-factor and evolution is proposed. Optomechanical changes have been considered as the resultant of intrinsic (e.g. fiber elastic modulus) and gross tissue mechanics (extracellular matrix (ECM)) in maturing scars. Non-invasive optomechanics supported with microscopic findings reveal scars cross-sectional self-organizing di-fork architecture. Dual-compartment heterogeneity of di-fork exhibits stress-evading features with a dichotomy in inhabitant cellular stress-fiber distributions. This differential interactivity of scar with adjoining tissues reflects its architectural intelligence to compensate tissue loss (hypodermis/muscle) by assembling into a di-fork. Gradual establishment of baseline shifted lasting mechanobiological steady-state, later in scarring, expose scar as an alternate stable state within the skin. Significance StatementWound repair in mammals, predominantly culminates into function compromising scar that is occasionally fatal in vital organs. How the biological system often adopts scarring over a restorative regeneration is yet a conundrum. Wound and ambient mechanics play a pivotal role in deciding the healing fate. SS-OCT is hence demonstrated here as a non-invasive window to such mechanical manifestations during skin wound healing. This exposed gradual emergence of temporally maintained and stress-resilient di-fork architecture of the scar with differential neighborhood interfaces. Accommodation of such an alternate self-organizing steady-state of scar sheds light on its sustenance and paradoxical selection.

biophysics↗

bioRxiv2017

Mechanical mapping of spinal cord development and repair in living zebrafish larvae using Brillouin microscopy

The mechanical properties of biological tissues are increasingly recognized as important factors in developmental and pathological processes. Most existing mechanical measurement techniques either necessitate destruction of the tissue for access or provide insufficient spatial resolution. Here, we show for the first time a systematic application of confocal Brillouin microscopy to quantitatively map the mechanical properties of spinal cord tissues during biologically relevant processes in a contact-free and non-destructive manner. Living zebrafish larvae were mechanically imaged in all anatomical planes, during development and after spinal cord injury. These experiments revealed that Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animals natural development. The Brillouin shift within the spinal cord increased during development and transiently decreased during the repair processes following spinal cord transection. By taking into account the refractive index distribution, we explicitly determined the apparent longitudinal modulus and viscosity of different larval zebrafish tissues. Importantly, mechanical properties differed between tissues in situ and in excised slices. The presented work constitutes the first step towards an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a methodical basis to identify key determinants of mechanical tissue properties and allows to test their relative importance in combination with biochemical and genetic factors during developmental and regenerative processes.

biophysics↗

bioRxiv2017

Topology and cooperative stability: the two master regulators of protein half-life in the cell

In a quest for finding additional structural constraints, apart from disordered segments, regulating protein half-life in the cell (and during evolution), here we recognize and assess the influence of native topology of biological proteins and their sequestration into multimeric complexes. Native topology acts as a molecular marker of proteins mechanical resistance and consequently captures their half-life variations on genome-scale, irrespective of the enormous sequence, structural and functional diversity of the proteins. Cooperative stability (slower degradation upon sequestration into complexes) is a master regulator of oligomeric protein half-life that involves at least three mechanisms. (i) Association with multiple complexes results longer protein half-life; (ii) hierarchy of complex self-assembly involves short-living proteins binding late in the assembly order and (iii) binding with larger buried surface area leads to slower subunit dissociation and thereby longer half-life. Altered half-lives of paralog proteins refer to their structural divergence and oligomerization with non-identical set of complexes.

biophysics↗

bioRxiv2017

Quantitation of brain tumour microstructure response to Temozolomide therapy using non-invasive VERDICT MRI

There has been slow progress in the development of new therapeutic strategies for treating brain tumours, partly because assessment of treatment response is difficult and largely reliant on simple bi-dimensional measurements of MRI contrast-enhancing regions. Hence, there is a clinical need to develop improved imaging techniques for monitoring treatment response. In this study, we evaluate VERDICT (Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumors) MRI in mouse glioblastomas for the quantification of tumour microstructure and assessment of response to Temozolomide (TMZ) chemotherapy, and, we investigate the feasibility of applying VERDICT MRI in a range of human gliomas. VERDICT MRI detected response to TMZ earlier than structural and apparent diffusion coefficient (ADC) measurements. A significant reduction in the cell radius parameter was detected three days earlier than ADC and six days earlier than structural MRI. Histological analysis showed the same trend as VERDICT of decreased intracellular volume fraction in the TMZ-treated mice. Vascular volume fraction was not altered by TMZ, which was consistent with optical projection tomography measurements. In patients, glioblastoma compartmental volume fractions showed good agreement with mouse glioblastoma parameters. The VERDICT parameters varied across the human gliomas, with raised intracellular volume fraction in the oligodendrogliomas and elevated cell radius in both low-grade tumours subtypes. In conclusion, our results suggest that VERDICT MRI is more sensitive at detecting TMZ response than structural or ADC measurements. In patients, VERDICT is feasible within clinical scan times, and performed best at characterising glioblastoma. Further optimisation should improve assessment of different glioma subtypes.

biophysics↗

bioRxiv2017

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. AcrB of Escherichia coli is a paradigm model of these polyspecific transporters. The molecular determinants and the energetics of the functional rotation mechanism proposed for the export of substrates by this protein have not yet been unveiled. To this aim, we implemented an original protocol that allows mimicking substrate transport in silico. We show that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the surface of the substrate transport channel. This, in turn, allows for a fairly constant hydration of the substrate that facilitates its diffusion. Our findings reveal a new molecular mechanism of transport in polyspecific systems, whereby waters contribute by screening potentially strong substrate-protein interactions. The mechanistic understanding of a fundamental process related to multi-drug transport provided here could help rationalizing the behavior of other polyspecific systems.

biophysics↗

bioRxiv2017

Single-cell analysis of early antiviral gene expression reveals a determinant of stochastic IFNB1 expression

RIG-I-like receptors (RLRs) are cytoplasmic sensors of viral RNA that trigger the signaling cascade that leads to type I interferon (IFN) production. Transcriptional induction of RLRs by IFN is believed to play the role of positive feedback to further amplify viral sensing. We found that RLRs and several other IFN-stimulated genes (ISGs) are induced early in viral infection independent of IFN. Expression of these early ISGs requires IRF3/IRF7 and is highly correlated amongst them. Simultaneous detection of mRNA of IFNB1, viral replicase, and ISGs revealed distinct populations of IFNB1 expressing and non-expressing cells which are highly correlated with the levels of early ISGs but are uncorrelated with IFN-dependent ISGs and viral gene expression. Individual expression of RLRs made IFNB1 expression more robust and earlier, suggesting a causal relation between levels of RLR and induction of IFN.

biophysics↗

bioRxiv2017

Structure-kinetic-thermodynamic relationships for peptide secondary structure formation identified from transition network properties

Coarse-grained molecular simulation models have provided immense, often general, insight into the complex behavior of condensed-phase systems, but suffer from a lost connection to the true dynamical properties of the underlying system. In general, the physics that is built into a model shapes the free-energy landscape, restricting the attainable static and kinetic properties. In this work, we perform a detailed investigation into the property interrelationships resulting from these restrictions, for a representative system of the helix-coil transition. Inspired by high-throughput studies, we systematically vary force-field parameters and monitor their structural, kinetic, and thermodynamic properties. The focus of our investigation is a simple coarse-grained model, which accurately represents the underlying structural ensemble, i.e., effectively avoids sterically-forbidden configurations. As a result of this built-in physics, we observe a rather large restriction in the topology of the networks characterizing the simulation kinetics. When screening across force-field parameters, we find that structurally-accurate models also best reproduce the kinetics, suggesting structural-kinetic relationships for these models. Additionally, an investigation into thermodynamic properties reveals a link between the cooperativity of the transition and the network topology at a single reference temperature.

biophysics↗

bioRxiv2017

Evidence of sinks and sources in the PLC activated PIP2 cycle

In many eukaryotic signalling cascades, receptor mediated phospholipase C (PLC) activity triggers phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis leading to information transfer in cells. Coupled with PLC activation is a sequence of reactions spread across multiple compartments by which PIP2 is resynthesized, a process essential to maintain PIP2 levels and support sustained PLC signalling. The biochemical strategies to co-ordinate these reactions and support PIP2 levels have remained poorly understood. In particular, the question of whether the PIP2 cycle is a closed cycle with no net addition or loss of metabolites has not been addressed. Using mathematical modelling approaches, we find that a closed PIP2 cycle cannot explain experimentally observed changes in the metabolic intermediates when changing enzyme activities in the PIP2 cycle. Thus, we propose that the PIP2 cycle likely includes at least one metabolic source and one sink whose net activity results in the experimentally observed regulation of this key signalling pathway.

biophysics↗