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bioRxiv2018

NMR Protein Structure Determination via Local Conformational Mapping

The ability of proteins to adopt multiple conformational states is essential to their function and elucidating the details of such diversity under physiological conditions has been a major challenge. Here we present a generalized method for mapping protein population landscapes by NMR spectroscopy. Experimental NOESY spectra are directly compared to a set of expectation spectra back-calculated across an arbitrary conformational space. Signal decomposition of the experimental spectrum then directly yields the relative populations of local conformational microstates. In this way, averaged descriptions of conformation can be eliminated. As the method quantitatively compares experimental and expectation spectra, it inherently delivers an R-factor expressing how well structural models explain the input data. We demonstrate that our method extracts sufficient information from a single 3D NOESY experiment to perform initial model building, refinement and validation, thus offering a complete de novo structure determination protocol.

biophysics↗

bioRxiv2018

Molecular dynamics ensemble refinement of the heterogeneous native state of NCBD using chemical shifts and NOEs

Many proteins display complex dynamical properties that are often intimately linked to their biological functions. As the native state of a protein is best described as an ensemble of confor-mations, it is important to be able to generate models of native state ensembles with high accuracy. Due to limitations in sampling efficiency and force field accuracy it is, however, challenging to obtain accurate ensembles of protein conformations by the use of molecular simulations alone. Here we show that dynamic ensemble refinement, which combines an accurate atomistic force field with commonly available nuclear magnetic resonance (NMR) chemical shifts and NOEs, can provide a detailed and accurate description of the conformational ensemble of the native state of a highly dynamic protein. As both NOEs and chemical shifts are averaged on timescales up to milliseconds, the resulting ensembles reflect the structural heterogeneity that goes beyond that probed e.g. by NMR relaxation order parameters. We selected the small protein domain NCBD as object of our study since this protein, which has been characterized experimentally in substantial detail, displays a rich and complex dynamical behaviour. In particular, the protein has been described as having a molten-globule like structure, but with a relatively rigid core. Our approach allowed us to describe the conformational dynamics of NCBD in solution, and to probe the structural heterogeneity resulting from both short- and long-time-scale dynamics by the calculation of order parameters on different time scales. These results illustrate the usefulness of our approach since they show that NCBD is rather rigid on the nanosecond timescale, but interconverts within a broader ensemble on longer timescales, thus enabling the derivation of a coherent set of conclusions from various NMR experiments on this protein, which could otherwise appear in contradiction with each other.

biophysics↗

bioRxiv2018

Recruitment of Two Dyneins to an mRNA-Dependent Bicaudal D Transport Complex

We investigated the role of binding partners of full-length Drosophila Bicaudal D (BicD) in the activation of dynein-dynactin motility for mRNA transport on microtubules. In single-molecule assays, full-length BicD robustly activated dynein-dynactin only when both the mRNA binding protein Egalitarian (Egl), and K10 mRNA cargo were present. Electron microscopy showed that both Egl and mRNA were needed to disrupt an auto-inhibited, looped BicD conformation that sterically prevents dynein-dynactin binding. In vitro reconstituted messenger ribonucleoprotein (mRNP) complexes with two Egl molecules showed faster speeds and longer run lengths than mRNPs with one Egl, suggesting that cargo binding enhances dynein recruitment. Labeled dynein showed that BicD can recruit two dimeric dyneins to the mRNP, resulting in faster speeds and longer run lengths than with one dynein. The fully reconstituted mRNP provides a model for understanding how adaptor proteins and cargo cooperate to confer optimal transport properties to a dynein-driven transport complex.

biophysics↗

bioRxiv2018

Stochastic models of cell invasion with fluorescent cell cycle indicators

Fluorescent cell cycle labelling in cell biology experiments provides real time information about the location of individual cells, as well as the phase of the cell cycle of individual cells. We develop a stochastic, lattice-based random walk model of a two-dimensional scratch assay where the total population is composed of three distinct subpopulations which we visualise as red, yellow and green subpopulations. Our model mimics FUCCI technology in which cells in the G1 phase of the cell cycle fluoresce red, cells in the early S phase fluoresce yellow, and cells in the S/G2/M phase fluoresce green. The model is an exclusion process so that any potential motility or proliferation event that would place an agent on an occupied lattice site is aborted. Using experimental images and previous experimental measurements, we explain how to apply the stochastic model to simulate a scratch assay initialised with a low to moderate density monolayer of human melanoma cell line. We obtain additional mathematical insight by deriving an approximate partial differential equation (PDE) description of the stochastic model, leading to a novel system of three coupled nonlinear reaction diffusion equations. Comparing averaged simulation data with the solution of the continuum limit model confirms that the PDE description is accurate for biologically-relevant parameter combinations.

biophysics↗

bioRxiv2018

Stimulus-responsive self-assembly of enzymatic fractal structures by computational design

Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns at increasing length scales in fractal-shaped branched objects, e.g., trees, lungs, and sponges, results in high effective surface areas, and provides key functional advantages, e.g., for molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies will provide access to novel classes of functional biomaterials for wide ranging applications. Here, we describe a modular, multi-scale computational design method for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Computationally-guided atomic-resolution modeling of fusions of symmetric, oligomeric proteins with Src homology 2 (SH2) binding domain and its phosphorylatable ligand peptide was used to design iterative branching leading to fractal-like assembly formation by enzymes of the atrazine degradation pathway. Structural characterization using various microscopy techniques and Cryo-electron tomography revealed a variety of dendritic, hyperbranched, and sponge-like topologies which are self-similar over three decades ([~]10nm-10m) of length scale, in agreement with models from multi-scale computational simulations. We demonstrate control over mesoscale topology (by linker design), formation dynamics, and functional enhancements due to dynamic multi-component assemblies constructed with three atrazine degradation pathway enzymes. The described design method should enable the construction of a variety of novel, spatiotemporally responsive catalytic biomaterials featuring fractal topologies.

biophysics↗

bioRxiv2018

Cryo-EM visualization of an active high open probability CFTR ion channel

The Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR with very high channel open probability, confirmed after reconstitution into proteoliposomes. The unique repositioning of the TMHs and R domain density that we observe provide insights into the structural transition between active and inactive functional states of CFTR.\n\nHighlightsO_LIStructures of thermostabilized avian CFTR in dephosphorylated or phosphorylated forms at 4.3 [A] and 6.6 [A] resolution, respectively.\nC_LIO_LIConformational differences of transmembrane helices 7 & 8 compared to zebra fish and human CFTR structures reveal an extracellular vestibule that may provide anion access to the pore.\nC_LIO_LIR-domain density appears to \"plug\" the intercellular vestibule in the dephosphorylated avian CFTR cryo-EM map.\nC_LI

biophysics↗

bioRxiv2018

Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

In Krammers theory, stiffness and dissipation coefficient of a protein determine the rate of their conformational change. Using atomic force microscope, it is possible to measure viscoelasticity of a single protein, wherein its dissipative and elastic nature is directly and independently measured. Such measurements are performed, either by measuring the thermal fluctuations of the protein held under a constant force, or by providing small modulations to the protein by dithering the cantilever and measuring its response. In small amplitude approximation, where dither amplitude is comparable to persistence length of polymers, it is possible to measure the proteins viscoelastic response accurately. We measured dissipation in I27 at extremely low pulling speeds ([~] 50 nm/s) and low dither frequencies ([~]100 Hz). At these experimental parameters the dissipation is found to be [~]10-5 kg/s, well above the detection limit of conventional AFM and upper limit predicted by Benedetti et al. Our stiffness data clearly reveals unfolding intermediate of titins individual immunoglobulin units. The intermediate is elongation of folded domains by [~]8 [A], wherein two hydrogen bonds are broken between beta sheets. It was possible to measure this elongation in our experiments. The directly measured internal friction of unfolded polymer chain shows a scaling with tension on the chain. The measurements show that it is possible to measure internal friction in single molecules unambiguously using small amplitude AFM. It suggests that systematic experiments to unravel the relation between directly measured internal friction and folding rates of proteins are possible.

biophysics↗

bioRxiv2018

Comparison between O and OH intermediates of cytochrome c oxidase studied by FTIR spectroscopy.

Cytochrome c oxidase is terminal enzyme in the respiratory chain of mitochondria and many aerobic bacteria. It catalyzes reduction of oxygen to water. During its catalysis, CcO proceeds through several quite stable intermediates (R, A, PR/M, O/OH, E/EH). This work is concentrated on the elucidation of the differences between structures of oxidized intermediates O and OH in different CcO variants and at different pH values. Oxidized intermediates of wild type and mutated CcO from Paracoccus denitrificans were studied by means of static and time-resolved Fourier-transform infrared spectroscopy in acidic and alkaline conditions in the infrared region 1800-1000 cm-1. No reasonable differences were found between all variants in these conditions, and in this spectral region. This finding means that the binuclear center of oxygen reduction keeps a very similar structure and holds the same ligands in the studied conditions. The further investigation in search of differences should be performed in the 4000-2000 cm-1 IR region where water ligands absorb.\n\nAbbreviations

biophysics↗

bioRxiv2018

A Hidden Markov Model for Detecting Confinement in Single Particle Tracking Trajectories

State-of-the-art single particle tracking (SPT) techniques can generate long trajectories with high temporal and spatial resolution. This offers the possibility of mechanistically interpreting particle movements and behaviour in membranes. To this end, a number of statistical techniques have been developed that partition SPT trajectories into states with distinct diffusion signatures, allowing a statistical analysis of diffusion state dynamics and switching behaviour. Here we develop a confinement model, within a hidden Markov framework, that switches between phases of free diffusion, and confinement in a harmonic potential well. By using a Markov chain Monte Carlo (MCMC) algorithm to fit this model, automated partitioning of individual SPT trajectories into these two phases is achieved, which allows us to analyse confinement events. We demonstrate the utility of this algorithm on a previously published dataset, where gold nanoparticle (AuNP) tagged GM1 lipids were tracked in model membranes. We performed a comprehensive analysis of confinement events, demonstrating that there is heterogeneity in the lifetime, shape, and size of events, with confinement size and shape being highly conserved within trajectories. Our observations suggest that heterogeneity in confinement events is caused by both individual nanoparticle characteristics and the binding site environment. The individual nanoparticle heterogeneity ultimately limits the ability of iSCAT to resolve molecular dynamics to the order of the tag size; homogeneous tags could potentially allow the resolution to be taken below this limit by deconvolution methods. In a wider context, the presented harmonic potential well confinement model has the potential to detect and characterise a wide variety of biological phenomena, such as hop diffusion, receptor clustering, and lipid rafts.

biophysics↗

bioRxiv2018

Adhesion of active cytoskeletal vesicles

Regulation of adhesion is a ubiquitous feature of living cells, observed during processes such as motility, antigen recognition or rigidity sensing. At the molecular scale, a myriad of mechanisms are necessary to recruit and activate the essential proteins, while at the cellular scale efficient regulation of adhesion relies on the cells ability to adapt its global shape. To understand the role of shape remodeling during adhesion, we use a synthetic biology approach to design a minimal model, starting with a limited number of building blocks. We assemble cytoskeletal vesicles whose size, reduced volume, and cytoskeleton contractility can be independently tuned. We are able to show that these cytoskeletal vesicles can sustain strong adhesion to solid substrates only if molecular motors are able to actively remodel the actin cortex. When the cytoskeletal vesicles are deformed under hypertonic osmotic pressure, they develop a crumpled geometry with huge deformations. In the presence of molecular motors, these deformations are dynamic in nature and can compensate for an absence of excess membrane area needed for adhesion to take place. When the cytoskeletal deformations are able to compensate for lack of excess membrane area, the cytoskeletal vesicles are able to attach to the rigid glass surfaces even under strong adhesive forces. The balance of deformability and adhesion strength is identified to be key to enable cytoskeletal vesicles to adhere to solid substrates.

biophysics↗

bioRxiv2018

A strategy to study intrinsically mixed folded proteins: The structure in solution of ataxin-3

It has increasingly become clear over the last two decades that proteins can contain both globular domains and intrinsically unfolded regions which both can contribute to function. While equally interesting, the disordered regions are difficult to study because they usually do not crystallize unless bound to partners and are not easily amenable to cryo-electron microscopy studies. Nuclear magnetic resonance spectroscopy remains the best technique to capture the structural features of intrinsically mixed folded proteins and describe their dynamics. These studies rely on the successful assignment of the spectrum, task not easy per se given the limited spread of the resonances of the disordered residues. Here, we describe assignment of the spectrum of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We used a 42 kDa construct containing a globular N-terminal josephin domain and a C-terminal tail which comprises thirteen polyglutamine repeats within a low-complexity region. We developed a strategy which allowed us to achieve 87% assignment of the spectrum. We show that the C-terminal tail is flexible with extended helical regions and interacts only marginally with the rest of the protein. We could also, for the first time, deduce the structure of the polyglutamine repeats within the context of the full-length protein and show that it has a strong helical propensity stabilized by the preceding region.

biophysics↗

bioRxiv2018

Lipid droplet phase transition in freezing cat embryos and oocytes probed by Raman spectroscopy

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One challenging limitation of this technology is the cell damage during freezing associated with the intracellular lipid droplets. We exploit a Raman spectroscopy to investigate the freezing of cumulus-oocyte complexes, mature oocytes and early embryos of a domestic cat. All these cells are rich in lipids. The degree of lipid unsaturation, lipid phase transition from liquid-like disordered to solid-like ordered state (main transition) and triglyceride polymorphic state are studied. For all cells examined, the average degree of lipid unsaturation is estimated about 1.3 (with {+/-}20 % deviation) double bonds per acyl chain. The onset of the main lipid phase transition occurs in a temperature range from -10 to +4 {degrees}C and does not depend significantly on the cell type. It is found that lipid droplets in cumulus-oocyte complexes undergo an abrupt lipid crystallization, which not completely correlate with the ordering of lipid molecule acyl chains. In the case of mature oocytes and early embryos obtained in vitro from cumulus-oocyte complexes, the lipid phase transition is broadened. In frozen state lipid droplets inside the cumulus-oocyte complexes have higher content of triglyceride polymorphic {beta} and {beta}' phases ([~]66%) than it is estimated for the mature oocytes and the early embryos ([~]50%). For the first time, to our knowledge, temperature evolution of lipid droplets phase state is examined. Raman spectroscopy is proved as a prospective tool for in situ monitoring of lipid phase state in single embryo/oocyte during freezing.

biophysics↗

bioRxiv2018

The Structural Basis of IKs Ion-Channel Activation: Mechanistic Insights from Molecular Simulations

Relating ion-channel (iCh) structural dynamics to physiological function remains a challenge. Current experimental and computational techniques have limited ability to explore this relationship in atomistic detail over physiological timescales. A framework associating iCh structure to function is necessary for elucidating normal and disease mechanisms. We formulated a modeling schema that overcomes the limitations of current methods through applications of Artificial Intelligence Machine Learning (ML). Using this approach, we studied molecular processes that underlie human IKs voltage mediated gating. IKs malfunction underlies many debilitating and life-threatening diseases. Molecular components of IKs that underlie its electrophysiological function include KCNQ1 (pore forming tetramer) and KCNE1 (auxiliary subunit). Simulations, using the IKs structure-function model, reproduced experimentally recorded saturation of gating charge displacement at positive membrane voltages, two-step voltage sensor (VS) movement shown by fluorescence, iCh gating statistics, and current-voltage (I-V) relationship. New mechanistic insights include - (1) pore energy profile determines iCh subconductance (SC), (2) entire protein structure, not limited to the pore, contributes to pore energy and channel SC, (3) interactions with KCNE1 result in two distinct VS movements, causing gating charge saturation at positive membrane voltages and current activation delay, and (4) flexible coupling between VS and pore permits pore opening at lower VS positions, resulting in sequential gating. The new modeling approach is applicable to atomistic scale studies of other proteins on timescales of physiological function.

biophysics↗

bioRxiv2018

Mathematical modeling and simulations for large-strain J-shaped diagrams of soft biological tissues

Herein, we study stress-strain diagrams of soft biological tissues such as animal skin, muscles and arteries by Finsler geometry (FG) modeling. The stress-strain diagram of these biological materials is always J-shaped and is composed of toe, heel, linear and failure regions. In the toe region, the stress is zero, and the length of this zero-stress region becomes very large ([~=] 150%) in, for example, certain arteries. In this paper, we study long-toe diagrams using two-dimensional (2D) and 3D FG modeling techniques and Monte Carlo (MC) simulations. We find that except for the failure region, large-strain J-shaped diagrams are successfully reproduced by the FG models. This implies that the complex J-shaped curves originate from the interaction between the directional and positional degrees of freedom of polymeric molecules, as implemented in the FG model.

biophysics↗

bioRxiv2018

Effect of chromatin flexibility on diffusive loop extrusion via molecular slip-links

We use Brownian dynamics simulations to study the formation of chromatin loops through diffusive sliding of molecular slip links, mimicking the behaviour of cohesin-like molecules. We recently proposed that diffusive sliding is sufficient to explain the extrusion of chromatin loops of hundreds of kilo-base-pairs (kbp), which may then be stabilised by interactions between cohesin and CTCF proteins. Here we show that the elasticity of the chromatin fibre strongly affects this dynamical process, and find that diffusive loop extrusion is more efficient on stiffer chromatin regions. Efficiency is also enhanced if cohesin loading sites are close to regions where CTCF is bound. In light of the heterogeneous physical properties of eukaryotic chromatin, we suggest that our results should be relevant to the looping and organisation of interphase chromosomes in vivo.

biophysics↗

bioRxiv2018

A fluorescence correlation spectrometer for measurements in cuvettes

We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single molecule measurements on samples inside regular cuvettes. We built this by using an Extra Long Working Distance (ELWD), 0.7 NA, air objective with working distance > 1.8 mm. We have achieved counts per molecule > 44 kHz, diffusion time < 64 s for rhodamine B in aqueous buffer and a confocal volume < 2 fl. The cuvette-FCS can be used for measurements over a wide range of temperature that is beyond the range permitted in the microscope-based FCS. Finally, we demonstrate that cuvette-FCS can be coupled to automatic titrators to study urea dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette-FCS in the experiments that are regularly performed in fluorimeters but are generally avoided in microscope-based FCS.

biophysics↗

bioRxiv2018

Fast Parallel Algorithm for Large Fractal Kinetic Models with Diffusion

Chemical kinetic simulations are usually based on the law of mass action that applies to behavior of particles in solution. Molecular interactions in a crowded medium as in a cell, however, are not easily described by such conventional mathematical treatment. Fractal kinetics is emerging as a novel method for simulating kinetic reactions in such an environment. To date, there has not been a fast, efficient, and, more importantly, parallel algorithm for such computations. Here, we present an algorithm with several novel features for simulating large (with respect to size and time scale) fractal kinetic models. We applied the fractal kinetic technique and our algorithm to a canonical substrate-enzyme model with explicit phase-separation in the product, and achieved a speed-up of up to 8 times over previous results with reasonably tight bounds on the accuracy of the simulation. We anticipate that this technique and algorithm will have important applications to simulation of intra-cell biochemical reactions with complex dynamic behavior.

biophysics↗

bioRxiv2018

Na/K pump: Single-channel vs. Two-access-channel

We re-studied the dialyzed Na/K pumps, the experimental foundation for the two-access-channel model. We found that the charge-movement pump currents, the major evidence for the two-access-channel, are only observed in certain situations. Once the stimulation pulse is high enough at low ionic concentration gradient, the backward pump currents disappeared. The two-directional charge movement pump currents become uni-directional pump currents showing that ions have passed through the channel across the cell membrane.\n\nA modified single-channel configuration is introduced to explain the pump currents as two-way charge movement current and the one-way current. The negatively charged amino acids deeply inside the pump channel constitute a spatial array that function as a collimator to align the moving ions in the channel lumen and an energy-well for the moving cations. If the stimulation pulse cannot drive ions to overcome the ionic concentration gradient, the ions will be entrapped into the energy-well as if the pump channel is obstructed showing two separated access-channels. Once the stimulation pulse is high enough to drive ions overcoming the ionic concentration gradient, ions will pass through the channel across the cell membrane so that Na/K pumps exhibit a single channel configuration.

biophysics↗