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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 595 records · Page 33Linked to original sources

bioRxiv2016

Laurdan and di-4-ANEPPDHQ probe different properties of the membrane

Lipid packing is a crucial feature of cellular membranes. Quantitative analysis of membrane lipid packing can be achieved using polarity sensitive probes whose emission spectrum depends on the lipid packing. However, detailed insight into the exact mechanism that causes the spectral shift is essential to interpret the data correctly. Here, we analysed frequently used polarity sensitive probes, Laurdan and di-4-ANEPPDHQ, to test whether the underlying physical mechanisms of their spectral shift is the same, thus whether they report on the same physico-chemical properties of the cell membrane. Their steady-state spectra as well as time-resolved emission spectra in solvents and model membranes showed that they probe different properties of the lipid membrane. Our findings are important for the application of these dyes in cell biology.

Biophysics↗

bioRxiv2016

Lessons learned from comparing molecular dynamics engines on the SAMPL5 dataset

We describe our efforts to prepare common starting structures and models for the SAMPL5 blind prediction challenge. We generated the starting input files and single configuration potential energies for the host-guest in the SAMPL5 blind prediction challenge for the GROMACS, AMBER, LAMMPS, DESMOND and CHARMM molecular simulation programs. All conversions were fully automated from the originally prepared AMBER input files using a combination of the ParmEd and InterMol conversion programs.\n\nWe find that the energy calculations for all molecular dynamics engines for this molecular set agree to a better than 0.1% relative absolute energy for all energy components, and in most cases an order of magnitude better, when reasonable choices are made for different cutoff parameters. However, there are some surprising sources of statistically significant differences. Most importantly, different choices of Coulombs constant between programs are one of the largest sources of discrepancies in energies. We discuss the measures required to get good agreement in the energies for equivalent starting configurations between the simulation programs, and the energy differences that occur when simulations are run with program-specific default simulation parameter values. Finally, we discuss what was required to automate this conversion and comparison.

Biophysics↗

bioRxiv2016

Logistic proliferation of cells in scratch assays is delayed

Scratch assays are used to study how a population of cells re-colonises a vacant region on a two-dimensional substrate after a cell mono-layer is scratched. These experiments are used in many applications including drug design for the treatment of cancer and chronic wounds. To provide insights into the mechanisms that drive scratch assays, solutions of continuum reaction-diffusion models have been calibrated to data from scratch as-says. These models typically include a logistic source term to model carrying capacity-limited proliferation, however the choice of using a logistic source term is often made without examining whether it is valid. Here we study the proliferation of PC-3 prostate cancer cells in a scratch assay, and we focus on the proliferation of these cells far away from the scratch. All experimental results for the scratch assay are compared with equivalent results from a proliferation assay where the cell monolayer is not scratched. Visual inspection of the evolution of the cell density as a function of time reveals a series of sigmoid curves that could be naively calibrated to the solution of the logistic growth model. However, careful analysis of the per capita growth rate as a function of density reveals several key differences between the proliferation of cells in scratch and proliferation assays. The per capita growth rate in the proliferation assay decreases, approximately linearly, with density in the proliferation assay suggesting that the logistic growth model is valid for the entire duration of the proliferation assay. However, the per capita growth rate in the scratch assay increases with density when the density is sufficiently small in the scratch assay, suggesting that the logistic growth model does not apply. Instead, guided by data, we find that there are two phases of proliferation in a scratch assay. At short time we have a disturbance phase where proliferation is not logistic, and this is followed by a growth phase where proliferation appears to be logistic. Accounting for the differences in the growth and disturbance phase, we obtain biologically realistic estimates of the proliferation rate and carrying capacity density. In contrast, simply calibrating the solution of the logistic growth equation to all data from the scratch assays, we obtain an excellent match between the data and the model, but the parameter estimates vary wildly and are not biologically realistic. Overall our study shows that simply calibrating the solution of a continuum model to a scratch assay might produce misleading parameter estimates, and this issue can be resolved by making a distinction between the disturbance and growth phases. Repeating our procedure for other scratch assays will provide insight into the roles of the disturbance and growth phases for different cell lines and scratch assays performed on different substrates.

Biophysics↗

bioRxiv2016

Co-operation, competition and crowding: a discrete framework linking Allee kinetics, nonlinear diffusion, shocks and sharp-fronted travelling waves

AO_SCPLOWBSTRACTC_SCPLOWInvasion processes are ubiquitous throughout cell biology and ecology. During invasion, individuals can become isolated from the bulk population and behave differently. We present a discrete, exclusion-based description of the birth, death and movement of individuals. The model distinguishes between individuals that are part of, or are isolated from, the bulk population by imposing different rates of birth, death and movement. This enables the simulation of various co-operative or competitive mechanisms, where there is either a positive or negative benefit associated with being part of the bulk population, respectively. The mean-field approximation of the discrete process gives rise to 22 different classes of partial differential equation, which can include Allee kinetics and nonlinear diffusion. Here we examine the ability of each class of partial differential equation to support travelling wave solutions and interpret the long time behaviour in terms of the individual-level parameters. For the first time we show that the strong Allee effect and nonlinear diffusion can result in shock-fronted travelling waves. We also demonstrate how differences in group and individual motility rates can influence the persistence of a population and provide conditions for the successful invasion of a population.

Biophysics↗

bioRxiv2016

Real-time observation of replicative helicase assembly onto single-stranded DNA

Replicative helicases load onto DNA at the start of replication, and play a vital role by driving the replication fork forward. These helicases assemble into closed multimeric rings that need to encircle single-stranded (ss)DNA to be activated. Though helicase loading on substrates with accessible free ends has been well characterized for the T7 gp4 helicase, a model system for superfamily IV replicative helicases, the physiologically more relevant loading onto exposed ssDNA without free ends remains less well understood. Here, using a label-free assay that exploits changes in the DNA hairpin hopping dynamics to detect gp4 binding and activity, we characterize loading and activation of gp4 on exposed ssDNA without free ends, and find clear evidence of stepwise assembly of the helicase at the fork at physiologically relevant concentrations. The gradual loading onto ssDNA, rather than pre-forming in solution followed by spontaneous ring opening which appears favored at higher concentrations, suggests a new paradigm of stepwise assembly for the helicases in superfamily IV that do not require a separate loading enzyme.

Biophysics↗

bioRxiv2016

Spatially periodic calcium profiles around single channels

The concept of calcium nanodomains established around the sites of calcium entry into the cell is fundamental for mechanistic consideration of key physiological responses. It stems from linear models of calcium diffusion from single channel into the cytoplasm, but is only valid for calcium increases smaller than the concentration of calcium-binding species. Recent experiments indicate much higher calcium levels in the vicinity of channel exit that should cause buffer saturation. I here derive explicit solutions of respective non-linear reaction-diffusion problem and found dichotomous solution - for small fluxes the steady state calcium profiles have quasi-exponential form, whereas in the case of buffer saturation calcium distributions show spatial periodicity. These non-trivial and novel spatial calcium profiles are supported by Monte-Carlo simulations. Imaging of 1D- and radial distributions around single -synuclein channels measured in cell-free conditions supports the theory. I suggest that periodic patterns may arise under different physiological conditions and play specific role in cell physiology.

Biophysics↗

bioRxiv2016

Composition fluctuations in lipid bilayers

Lipid bilayers constitute the basis of biological membranes. Understanding lipid mixing and phase behavior can provide important insights into membrane lateral organization (the \"raft\" hypothesis). Here we investigate model lipid bilayers below and above their miscibility transition temperatures. Molecular dynamics simulations with the MARTINI coarse-grained force field are employed to model bilayers on a length scale approaching 100 nm and a time scale of tens of microseconds. Using a binary mixture of saturated and unsaturated lipids, and a ternary mixture of a saturated lipid, an unsaturated lipid and cholesterol we reproduce the coexistence of liquid-crystalline and gel, as well as liquid-ordered and liquid-disordered phases. By raising the temperature or adding hybrid lipids (with a saturated and an unsaturated chain), we induce a gradual transition from a two-phase to a one-phase state. We characterize the evolution of bilayer properties along this transition. Domains of coexisting phases change to dynamic heterogeneity with local ordering and compositional de-mixing. We analyze the structural and dynamic properties of domains, sizes and lifetimes of composition fluctuations, and calculate the in-plane structure factors.

Biophysics↗

bioRxiv2016

Unique properties of Zika NS2B-NS3pro complexes as decoded by experiments and MD simulations

Zika virus can be passed from a pregnant woman to her fetus, thus leading to birth defects including more than microcephaly. It has been recently estimated that one-third of the world population will be infected by Zika, but unfortunately no vaccine or medicine is available so far. Zika NS2B-NS3pro is essential for its replication and thus represents an attractive target for drug discovery/design. Here we characterized conformation, catalysis, inhibition and dynamics of linked and unlinked Zika NS2B-NS3pro complexes by both experiments and MD simulations. The results unveil the unique properties of Zika NS2B-NS3pro which are very different from Dengue one. Particularly, CD and NMR studies indicate that unlike Dengue, the C-terminal region of Zika NS2B with a significant sequence variation is highly disordered in the open conformation. Indeed, MD simulations reveal that up to 100 ns, the Dengue NS2B C-terminus constantly has close contacts with its NS3pro domain. By a sharp contrast, the Zika NS2B C-terminus loses the contacts with its NS3pro domain after 10 ns, further forming a short {beta}-sheet characteristic of the closed conformation at 30 ns. Furthermore, we found that a small molecule, previously identified as an active site inhibitor for other flaviviral NS2B-NS3pro, inhibited Zika NS2B-NS3pro potently in an allosteric manner. Our study provides the first insight into the dynamics of Zika NS2B-NS3pro and further deciphers that it is susceptible to allosteric inhibition, which thus bears critical implications for the future development of therapeutic allosteric inhibitors.

Biophysics↗

bioRxiv2016

A pipeline approach to single-particle processing in RELION

We describe the formal concept of a workflow to single-particle analysis of cryo-electron microscopy (cryo-EM) images in the RELION program. In this approach, the structure determination process is considered as a graph, where intermediate results in the form of images or metadata are the vertices, and different functionalities of the program are the edges. The new implementation automatically logs all user actions, facilitates file management and disk cleaning, and allows convenient browsing of a projects history. Moreover, new functionality to iteratively execute consecutive jobs allows on-the-fly image processing, which will lead to more efficient data acquisition by providing faster feedback on data quality. The possibility to exchange data processing procedures among users will con-tribute to the development of standardised image processing procedures, and hence increase accessibility for new users in this rapidly expanding field.

Biophysics↗

bioRxiv2016

Identification of a Zika NS2B-NS3pro pocket susceptible to allosteric inhibition by small molecules including qucertin rich in edible plants

It has been recently estimated that one-third of the world population will be infected by Zika virus, but unfortunately so far there is no vaccine or medicine available. In particular, the special concern on the vaccine treatment to Zika and Dengue arising from antibody-dependent enhancement strongly emphasizes the irreplaceable role of its NS2B-NS3 protease (NS2B-NS3pro) as a target for anti-Zika drug discovery/design due to its absolutely-essential role in viral replication. Very recently we identified two small molecules inhibit Zika NS2B-NS3pro in non-competitive mode, with Ki values of 0.57 and 2.02 {micro}M respective for p-Nitrophenyl-p-guanidino benzoate and qucertin. Here, by molecular docking, we show that although one is designed compound while another is a natural product, both molecules bind to the same pocket on the back of the substrate-binding pocket of Zika NS2B-NS3pro. As the two inhibitors fundamentally differ from cn-716, the only known peptidomimetic boronic acid inhibitor in both structure scaffolds and inhibitory modes, our discovery might open up a new avenue for the future development of allosteric inhibitors, which is highly demanded to achieve therapeutic inhibition of flaviviral NS2B-NS3pro complexes. Furthermore, as qucertin is abundant in many vegetables and fruits such caper, lovage, tea and red onion, our results should benefit the public to immediately fight Zika virus.

Biophysics↗

bioRxiv2016

Kinematics of the Lever Arm Swing in Myosin VI

Myosin VI (MVI) is the only known member of the myosin superfamily that, upon dimerization, walks processively towards the pointed end of the actin filament. The leading head of the dimer directs the trailing head forward with a power stroke, a conformational change of the motor domain exaggerated by the lever arm. Using a new coarse-grained model for the power stroke of a single MVI, we provide the molecular basis for its motility. We show that the power stroke occurs in two major steps: first, the motor domain attains the post-stroke conformation without directing the lever arm forward; second, the lever arm reaches the post-stroke orientation by undergoing a rotational diffusion. From the analysis of the trajectories, we discover that the potential that directs the rotating lever arm towards the post-stroke conformation is almost flat, implying that the lever arm rotation is mostly un-coupled from the motor domain. Because a backward load comparable with the largest inter-head tension in a MVI dimer prevents the rotation of the lever arm, our model suggests that the leading-head lever arm of a MVI dimer is uncoupled, in accord with the inference drawn from polarized Total Internal Reflection Fluorescence (polTIRF) experiments. Our simulations are in quantitative agreement with polTIRF experiments, which validates our structural insights. Finally, we discuss the implications of our model in explaining the broad step-size distribution of MVI stepping pattern, and we make testable predictions.

Biophysics↗

bioRxiv2016

A unified coarse-grained theory of bacterial physiology explains the relationship between cell size, growth rate and proteome composition under various growth limitations

Universal observations in Biology are sometimes described as "laws". In E. coli, experimental studies performed over the past six decades have revealed major growth laws relating ribosomal mass fraction and cell size to the growth rate. Because they formalize complex emerging principles in biology, growth laws have been instrumental in shaping our understanding of bacterial physiology. Here, we discovered a novel size law that connects cell size to the inverse of the metabolic proteome mass fraction and the active fraction of ribosomes. We used a simple whole-cell coarse-grained model of cell physiology that combines the proteome allocation theory and the structural model of cell division. The model captures all available experimental data connecting the cell proteome composition, ribosome activity, division size and growth rate in response to nutrient quality, antibiotic treatment and increased protein burden. Finally, a stochastic extension of the model explains non-trivial correlations observed in single cell experiments including the adder principle. This work provides a simple and robust theoretical framework for studying the fundamental principles of cell size determination in unicellular organisms.

Biophysics↗

bioRxiv2016

Hi-C-constrained physical models of human chromosomes recover functionally-related properties of genome organization

Combining genome-wide structural models with phenomenological data is at the forefront of efforts to understand the organizational principles regulating the human genome. Here, we use chromosome-chromosome contact data as knowledge- based constraints for large-scale three-dimensional models of the human diploid genome. The resulting models remain minimally entangled and acquire several functional features that are observed in vivo and that were never used as input for the model. We find, for instance, that gene-rich, active regions are drawn towards the nuclear center, while gene poor and lamina-associated domains are pushed to the periphery. These and other properties persist upon adding local contact constraints, suggesting their compatibility with non-local constraints for the genome organization. The results show that suitable combinations of data analysis and physical modelling can expose the unexpectedly rich functionally-related properties implicit in chromosome-chromosome contact data. Specific directions are suggested for further developments based on combining experimental data analysis and genomic structural modelling.

biophysics↗

bioRxiv2016

TatA complexes exhibit a marked change in organisation in response to expression of the TatBC complex

The twin arginine translocation (Tat) system is an integral membrane protein complex that accomplishes the remarkable feat of transporting large, fully-folded polypeptides across the inner membrane of bacteria, into the periplasm. In Escherichia coli Tat is comprised of three membrane proteins: TatA, TatB and TatC. How these proteins arrange themselves in the inner membrane to permit passage of Tat substrates, whilst maintaining membrane integrity, is still poorly understood. TatA is the most abundant component of this complex and facilitates assembly of the transport mechanism. We have utilised immunogold labelling in combination with array tomography to gain insight into the localisation and distribution of the TatA protein in E. coli cells. We show that TatA exhibits a uniform distribution throughout the inner membrane of E. coli and that altering the expression of TatBC shows a previously uncharacterised distribution of TatA in the inner membrane. Array tomography was used to provide our first insight into this altered distribution of TatA in 3D space, revealing that this protein forms linear clusters in the inner membrane of E. coli upon increased expression of TatBC. This is the first indication that TatA organisation in the inner membrane alters in response to changes in Tat subunit stoichiometry.\n\nSummary statementThe volumetric electron-microscopy technique, array tomography, revealed a novel distribution of TatA protein (from the twin arginine translocase complex), in Escherichia coli.

Biophysics↗

bioRxiv2016

A Probabilistic Approach to the Detection and Diagnosis of Obstructive Sleep Apnea and Its Environmental Factors Using Chest Movement Analysis, Recursive Noise Reduction and Machine Learning Algorithms.

1In a novel approach to diagnose Obstructive Sleep Apnea, electronic components, such as an Arduino Mega, a Bluetooth Transceiver, an accelerometer, and a air-quality sensor, were put together to create a wearable that would detect the frequency of apnea events, detect and diagnose the disorder, and sound an alarm when necessary. A primary consideration was to make the mechanism accessible and affordable, and in doing so, lower the cases of Obstructive Sleep Apnea that go undiagnosed due to the cost and inconvenience associated with the traditional diagnosis method--a Polysomnogram. Bluetooth capability was an additional consideration so that the device would transmit data directly to an android smartphone, eliminating the need for an additional output mechanism. The total cost of the device, quite surprisingly, did not exceed $30, and therein rendered the device an accessible, affordable mechanism for diagnosis. Tests of the device on diagnosed patients yielded data consistent with the diagnosis, with a few false positives as a result of the excessive sensitivity of the sensors.

biophysics↗

bioRxiv2016

DNA Methylation Landscape Reflects the Spatial Organization of Chromatin in Different Cells

The relation between DNA methylation and chromatin structure is still largely unknown. By analyzing a large set of sequencing data, we observed a long-range power law correlation of DNA methylation with cell-class-specific scaling exponents in the range of thousands to millions of base pairs. We showed such cell-class-specific scaling exponents are caused by different patchiness of DNA methylation in different cells. By modeling the chromatin structure using Hi-C data and mapping the methylation level onto the modeled structure, we demonstrated the patchiness of DNA methylation is related to chromatin structure. The scaling exponents of the power law correlation is thus a display of the spatial organization of chromatin. Besides, the local correlation of DNA methylation is associated with nucleosome positioning and different between partially-methylated-domain and non-partially-methylated-domain, suggesting their different chromatin structures at several nucleosomes level. Our study provides a novel view of the spatial organization of chromatin structure from a perspective of DNA methylation, in which both long-range and local correlations of DNA methylation along the genome reflect the spatial organization of chromatin.

biophysics↗

bioRxiv2016

Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin

Despite the small size the folding of Ubiquitin (Ub), which plays an indispensable role in targeting proteins for degradation and DNA damage response, is complex. A number of experiments on Ub folding have reached differing conclusions regarding the relation between collapse and folding, and whether intermediates are populated. In order to resolve these vexing issues, we elucidate the denaturant-dependent thermodynamics and kinetics of Ub folding in low and neutral pH as a function of Guanidinium chloride and Urea using coarse-grained molecular simulations. The changes in the fraction of the folded Ub, and the radius of gyration (Rg) as a function of the denaturant concentration, [C], are in quantitative agreement with experiments. Under conditions used in experiments, Rg of the unfolded state at neutral pH changes only by {approx} 17% as the [GdmCl] decreases from 6 M to 0 M. We predict that the extent of compaction of the unfolded state increases as temperature decreases. A two-dimensional folding landscape as a function of Rg and a measure of similarity to the folded state reveals unambiguously that the native state assembly is preceded by collapse, as discovered in fast mixing experiments on several proteins. Analyses of the folding trajectories, under mildly denaturing conditions ([GdmCl]=1.0M or [Urea]=1.0M), shows that Ub folds by collision between preformed secondary structural elements involving kinetic intermediates that are primarily stabilized by long-range contacts. Our work explains the results of Small Angle X-Ray Scattering (SAXS) experiments on Ub quantitatively, and establishes that evolved globular proteins are poised to collapse. In the process, we explain the discrepancy between SAXS and single molecule fluorescent resonant energy transfer (smFRET) experiments, which have arrived at a contradicting conclusion concerning the collapse of polypeptide chains.

biophysics↗

bioRxiv2016

Flexibility and design: conformational heterogeneity along the evolutionary trajectory of a redesigned ubiquitin

SummaryAlthough protein design has been used to introduce new functions, designed variants generally only function as well as natural proteins after rounds of laboratory evolution. One possibility for this pattern is that designed mutants frequently sample nonfunctional conformations. To test this idea, we exploited advances in multiconformer modeling of room temperature X-ray data collection on redesigned ubiquitin variants selected for increasing binding affinity to the deubiquitinase USP7. Initial core mutations disrupt natural packing and lead to increased flexibility. Additional, experimentally selected mutations quenched conformational heterogeneity through new stabilizing interactions. Stabilizing interactions, such as cation-pi stacking and ordered waters, which are not included in standard protein design energy functions, can create specific interactions that have long range effects on flexibility across the protein. Our results suggest that increasing flexibility may be a useful strategy to escape local minima during initial directed evolution and protein design steps when creating new functions.

biophysics↗