PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.31.231274v1?rss=1

Authors: Mickolajczyk, K. J., Shelton, P. M. M., Grasso, M., Cao, X., Warrington, S. R., Aher, A., Liu, S., Kapoor, T. M.

Abstract:

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.227959v1?rss=1

Authors: Rufa, D. A., Bruce Macdonald, H. E., Fass, J., Wieder, M., Grinaway, P. B., Roitberg, A. E., Isayev, O., Chodera, J. D.

Abstract:

Alchemical free energy methods with molecular mechanics (MM) force fields are now widely used in the prioritization of small molecules for synthesis in structure-enabled drug discovery projects because of their ability to deliver 1-2 kcal/mol accuracy in well-behaved protein-ligand systems. Surpassing this accuracy limit would significantly reduce the number of compounds that must be synthesized to achieve desired potencies and selectivities in drug design campaigns. However, MM force fields pose a challenge to achieving higher accuracy due to their inability to capture the intricate atomic interactions of the physical systems they model. A major limitation is the accuracy with which ligand intramolecular energetics---especially torsions---can be modeled, as poor modeling of torsional profiles and coupling with other valence degrees of freedom can have a significant impact on binding free energies. Here, we demonstrate how a new generation of hybrid machine learning / molecular mechanics (ML/MM) potentials can deliver significant accuracy improvements in modeling protein-ligand binding affinities. Using a nonequilibrium perturbation approach, we can correct a standard, GPU-accelerated MM alchemical free energy calculation in a simple post-processing step to efficiently recover ML/MM free energies and deliver a significant accuracy improvement with small additional computational effort. To demonstrate the utility of ML/MM free energy calculations, we apply this approach to a benchmark system for predicting kinase:inhibitor binding affinities---a congeneric ligand series for non-receptor tyrosine kinase TYK2 (Tyk2)---wherein state-of-the-art MM free energy calculations (with OPLS2.1) achieve inaccuracies of 0.93{+/-}0.12 kcal/mol in predicting absolute binding free energies. Applying an ML/MM hybrid potential based on the ANI2x ML model and AMBER14SB/TIP3P with the OpenFF 1.0.0 ("Parsley") small molecule force field as an MM model, we show that it is possible to significantly reduce the error in absolute binding free energies from 0.97 [95% CI: 0.68, 1.21] kcal/mol (MM) to 0.47 [95% CI: 0.31, 0.63] kcal/mol (ML/MM).

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.224972v1?rss=1

Authors: Malhotra, I., Oyarzun, B., Mognetti, B.

Abstract:

Nuclear molecules control the functional properties of the chromatin fiber by shaping its morphological properties. The biophysical mechanisms controlling how bridging molecules compactify the chromatin are a matter of debate. On the one side, bridging molecules could cross-link faraway sites and fold the fiber through the formation of loops. Interacting bridging molecules could also mediate long-range attractions by first tagging different locations of the fiber and then undergoing microphase separation. Using a coarse-grained model and Monte Carlo simulations, we study the conditions leading to compact configurations both for interacting and non-interacting bridging molecules. In the second case, we report on an unfolding transition at high densities of the bridging molecules. We clarify how this transition, which disappears for interacting bridging molecules, is universal and controlled by entropic terms. In general, chains are more compact in the case of interacting bridging molecules since, in this case, interactions are not valence-limited. However, this result is conditional on the ability of our simulation methodology to relax the system towards its ground state. In particular, we clarify how, unless using reaction dynamics that change the length of a loop in a single step, the system is prone to remain trapped in metastable, compact configurations featuring long loops.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.227199v1?rss=1

Authors: Zaichuk, T., Marko, J.

Abstract:

Cytosine methylated at the 5-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA, under physiologically relevant stretching forces and supercoiling densities. The stiffness and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226357v1?rss=1

Authors: Thiagarajan, R., Bhat, A., Salbreux, G., Inamdar, M. M., Riveline, D.

Abstract:

Epithelial cells flows are observed both in vivo and in vitro and are essential for morphogenesis. Here, we show that pulsatile flows involving local contraction and expansion of a tissue can arise in vitro in an epithelial monolayer of Madine Darby Canine Kidney (MDCK) cells. The strength of pulsation can be modulated through friction heterogeneity by observing the monolayer dynamics on micro-contact printed fibronectin grids with dimensions matching the length-scale of spontaneous oscillations. We also report pulsations by inducing wound closure in domains of similar size with micro-fabricated pillars. In contrast, strongly coherent flows can be induced by adding and washing out acto-myosin cytoskeleton inhibitors. To gain insight into the associated cellular mechanisms, we fluorescently label actin and myosin. We find that lamellipodia align with the direction of the flow, and tissue-scale myosin gradients arise during pulsations in wound-healing experiments. Pulsations and flows are recapitulated in silico by a vertex model with cell motility and polarisation dynamics. The nature of collective movements depends on the interplay between velocity alignment and random diffusion of cell polarisation. When they are comparable, a significant pulsatile flow emerges, whereas the tissue undergoes long-range flows when alignment dominates. We conjecture that the interplay between lamellipodial motile activity and cell polarization, with a possible additional role for tissue-scale myosin gradients, is at the origin of the pulsatile nature of the collective flow. Altogether, our study reveals that monolayer dynamics is dictated by simple rules of interaction at cellular levels which could be involved in morphogenesis.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.227322v1?rss=1

Authors: Xu, X., Gagne, D., Aramini, J. M., Gardner, K. H.

Abstract:

Proteins often interconvert between different conformations in ways critical to their function. However, manipulating the equilibrium positions and kinetics of such conformational transitions has been traditionally challenging. Pressure is an effective thermodynamic variable for favoring the population of high energy protein conformational states with smaller volumes, as elegantly demonstrated in its use for biophysical studies of unfolding transitions. Here, we investigate the pressure-dependent effects of the interconversion of two stably-folded conformations of the Y456T variant of the aryl hydrocarbon receptor nuclear translocator (ARNT) Period/ARNT/Single-minded PAS-B domain. We previously discovered that ARNT PAS-B Y456T spontaneously interconverts between two structures primarily differing by a three-residue {beta}-strand slip, inverting its topology in the process. This change collapses the internal cavities of the WT-like (WT) conformation of this protein as it adopts the slipped conformation (SLIP). Using pressure-NMR to conduct thermodynamic and kinetic analyses of the interconversion of ARNT PAS-B, we provide data that support two key predictions of this process: the SLIP conformation is both smaller and less compressible than the WT conformation, and the interconversion proceeds through a chiefly-unfolded intermediate state. We also find that the pressure-dependent NMR chemical shift changes and the residue-specific compressibility both predict which amino acid residues are near protein cavities. We demonstrate that pressure-NMR is a powerful approach for characterizing protein conformational switching and can provide unique information that is not easily accessible through other techniques.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226928v1?rss=1

Authors: Mioduszewski, Łukasz, Cieplak, M.

Abstract:

Wheat (Triticum spp.) gluten consists mainly of intrinsincally disordered storage proteins (glutenins and gliadins) that can form megadalton-sized networks. These networks are responsible for the unique viscoelastic properties of wheat dough and affect the quality of bread. These properties have not yet been studied by molecular level simulations. Here, we use a newly developed -C-based coarse-grained model to study [~] 4000 -residue systems. The corresponding time-dependent properties are studied through shear and axial deformations. We measure the response force to the deformation, the number of entanglements and cavities, the size of fluctuations, the number of the inter-chain bonds, etc. Glutenins are shown to influence the mechanics of gluten much more than gliadins. Our simulations are consistent with the existing ideas about gluten elasticity and emphasize the role of entanglements and hydrogen bonding. We also demonstrate that the storage proteins in maize and rice lead to weaker elasticity which points to the unique properties of wheat gluten.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.227231v1?rss=1

Authors: Huang, H., Chamness, L. M., Vanoye, C. G., Kuenze, G., Meiler, J., George, A. L., Schlebach, J. P., Sanders, C.

Abstract:

Gain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. We tested whether any of the known GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface: super-trafficking. We found that levels of R231C KCNQ1 in the plasma membrane are 5-fold higher than wild type KCNQ1. This arises from both enhanced translocon-mediated membrane integration of the S4 voltage-sensor helix and an energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 is constitutively active, but also revealed the single channel activity of this mutant to be only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the net effects of super-trafficking, reduced single channel activity, and constitutive channel activation. These investigations document membrane protein super-trafficking as a contributing mechanism to human disease.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226480v1?rss=1

Authors: LONATI, L., Barbieri, S., Guardamagna, I., Ottolenghi, A., Baiocco, G.

Abstract:

Cell cycle progression can be studied with computational models that allow to describe and predict its perturbation by agents as ionizing radiation or drugs. Such models can then be integrated in tools for pre-clinical/clinical use, e.g. to optimize kinetically-based administration protocols of radiation therapy and chemotherapy. We present a deterministic compartmental model, specifically reproducing how cells that survive radiation exposure are distributed in the cell cycle as a function of dose and time after exposure. Model compartments represent the four cell-cycle phases, as a fuction of DNA content and time. A system of differential equations, whose parameters represent transition rates, division rate and DNA synthesis rate, describes the temporal evolution. Initial model inputs are data from unexposed cells in exponential growth. Perturbation is implemented as an alteration of model parameters that allows to best reproduce cell-cycle profiles post-irradiation. The model is validated with dedicated in vitro measurements on human lung fibroblasts (IMR90). Cells were irradiated with 2 and 5 Gy with a Varian 6 MV Clinac at IRCCS Maugeri. Flow cytometry analysis was performed at the RadBioPhys Laboratory (University of Pavia), obtaining cell percentages in each of the four phases in all studied conditions up to 72 hours post-irradiation. Cells show early G2-phase block (increasing in duration as dose increases) and later G1-phase accumulation. For each condition, we identified the best sets of model parameters that lead to a good agreement between model and experimental data, varying transition rates from G1- to S- and from G2- to M-phase. This work offers a proof-of-concept validation of the new computational tool, opening to its future development and, in perspective, to its integration in a wider framework for clinical use.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.07.29.226761v1?rss=1

Authors: El-Baba, T. J., Lutomski, C. A., Kantsadi, A. L., Malla, T. R., John, T., Mikhailov, V., Bolla, J. R., Schofield, C. J., Zitzmann, N., Vakonakis, I., Robinson, C. V.

Abstract:

Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 {+/-} 0.03 {micro}M, revealing MPro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively flash-frozen as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

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