PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.296582v1?rss=1

Authors: Chen, Y., Matveev, V.

Abstract:

We examine closed-form approximations for the equilibrium Ca2+ concentration near a point Ca2+ source representing a Ca2+ channel, in the presence of a mobile Ca2+ buffer with 2:1 Ca2+ binding stoichiometry. We consider buffers with two Ca2+ binding sites activated in tandem and possessing distinct binding affinities and kinetics. This allows to model the impact on Ca2+ nanodomains of realistic endogenous Ca2+ buffers characterized by cooperative Ca2+ binding, such as calretinin. The approximations we present involve a combination or rational and exponential functions, whose parameters are constrained using the series interpolation method that we recently introduced for the case of 1:1 Ca2+ buffers. We conduct extensive parameter sensitivity analysis and show that the obtained closed-form approximations achieve reasonable qualitative accuracy for a wide range of buffer's Ca2+ binding properties and other relevant model parameters. In particular, the accuracy of the newly derived approximants exceeds that of the rapid buffering approximation in large portions of the relevant parameter space.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.296012v1?rss=1

Authors: Janecek, M., Kuhrova, P., Mlynsky, V., Otyepka, M., Sponer, J., Banas, P.

Abstract:

Representation of electrostatic interactions by a Coulombic pair-wise potential between atom-centered partial charges is a fundamental and crucial part of empirical force fields used in classical molecular dynamics simulations. The broad success of the AMBER force field family originates mainly from the restrained electrostatic potential (RESP) charge model, which derives partial charges to reproduce the electrostatic field around the molecules. However, description of the electrostatic potential around molecules by standard RESP may be biased for some types of molecules. In this study, we modified the RESP charge derivation model to improve its description of the electrostatic potential around molecules, and thus electrostatic interactions in the force field. In particular, we re-optimized the atomic radii for definition of the grid points around the molecule, redesigned the restraining scheme and included extra point charges. The RESP fitting was significantly improved for aromatic heterocyclic molecules. Thus, the suggested W-RESP(-EP) charge derivation model showed clear potential for improving the performance of the nucleic acid force fields, for which poor description of nonbonded interactions, such as underestimated base pairing, makes it difficult to describe the folding free energy landscape of small oligonucleotides.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.296111v1?rss=1

Authors: Erkip, A., Hacisuleyman, A., Erman, B., Erman, B.

Abstract:

We developed a Dynamic Gaussian Network Model to study perturbation and response in proteins. The model is based on the solution of the Langevin equation in the presence of noise and perturbation. A residue is perturbed periodically with a given frequency and the response of other residues is determined in terms of a storage and loss modulus of the protein. The amount of work lost upon periodic perturbation and the residues that contribute significantly to the lost work is determined. The model shows that perturbation introduces new dynamic correlations into the system with time delayed synchronous and asynchronous components. Residues whose perturbation induces large correlations in the protein and those that do not lead to correlations may be identified. The model is used to investigate the dynamic modulation of nanobodies. Despite its simplicity, the model explains several features of perturbation and response such as the role of loops and linkers in perturbation, dispersion of work of perturbation, and information transfer through preexisting pathways, all shown to be important factors in allostery.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.13.295345v1?rss=1

Authors: Iljina, M., Mazal, H., Goloubinoff, P., Riven, I., Haran, G.

Abstract:

ClpB is an ATP-dependent protein disaggregation machine that is activated on demand by co-chaperones and by aggregates caused by heat shock or mutations. The regulation of ClpB's function is critical, since its persistent activation is toxic in vivo. Each ClpB molecule is composed of an auxiliary N-terminal domain (NTD), an essential regulatory middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains that are responsible for ATP-fuelled substrate threading. The NTD is generally thought to serve as a substrate-binding domain, which is commonly considered to be dispensable for ClpB's activity, and is not well-characterized structurally due to its high mobility. Here we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB's NTD and reveal its involvement in novel allosteric interactions. We find that the NTD fluctuates on a microsecond timescale and, unexpectedly, shows little change in conformational dynamics upon binding of a substrate protein. During its fast motion, the NTD makes crucial contacts with the regulatory MD, directly affecting its conformational state and thereby influencing the overall ATPase and unfolding activity of this machine. Moreover, we also show that the NTD mediates signal transduction to the nucleotide-binding domains through conserved residues. The two regulatory pathways revealed here enable the NTD to suppress the MD in the absence of protein substrate, and to limit ATPase and disaggregation activities of ClpB. The use of multiple parallel allosteric pathways involving ultrafast domain motions might be common to AAA+ molecular machines to ensure their fast and reversible activation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.296756v1?rss=1

Authors: Budiardjo, S. J., Ikujuni, A. P., Firlar, E., Cordova, A., Kaelber, J., Slusky, J. S. G.

Abstract:

Overexpression of tripartite efflux pump systems in gram-negative bacteria are a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. Here, we develop a concentration-dependent folding protocol for refolding TolC, the outer membrane component of the antibiotic efflux pump from Escherichia coli. We show that by our method of re-folding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that a key factor in successful re-folding is a concentration dependence of TolC oligomerization. We extended the scheme to CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer-membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in re-folding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.296129v1?rss=1

Authors: Voegele, A., Sadi, M., O'Brien, D. P., Gehan, P., Raoux-Barbot, D., Davi, M., Hoss, S., Brule, S., Raynal, B., Weber, P., Mechaly, A., Haouz, A., Rodriguez, N., Vachette, P., Durand, D., Brier, S., Ladant, D., Chenal, A.

Abstract:

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.289330v1?rss=1

Authors: Vinje, J., Guadagno, N. A., Progida, C., Sikorski, P.

Abstract:

Cells in their natural environment are embedded in a complex surrounding consisting of biochemical and biomechanical cues directing cell properties and cell behaviour. Nonetheless, in vitro cell studies are typically performed on flat surfaces, with clear differences from the more complex situation cells experience in vivo. To increase the physiological relevance of these studies, a number of advanced cellular substrates for in vitro studies have been applied. One of these approaches include flat surfaces decorated with vertically aligned nanostructures. In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.14.295824v1?rss=1

Authors: Jack, A., Ferro, L. S., Trnka, M. J., Wehri, E., Nadgir, A., Costa, K., Schaletzky, J., Yildiz, A.

Abstract:

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with unstructured RNA, it forms mesh like-structures with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified an intrinsically-disordered region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. By screening 1,200 FDA approved drugs in vitro, we identified a kinase inhibitor nilotinib, which affects the morphology of N condensates in vitro and disrupts phase separation of the N protein in vivo. These results indicate that the N protein compartmentalizes viral RNA in infected cells through liquid-liquid phase separation, and this process can be disrupted by a possible drug candidate.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.12.286559v1?rss=1

Authors: Pan, H., Kaur, P., Liu, M., Xu, P., Mahn, C., Barnes, R., Tang, Q., Hao, P., Bhattaram, D., You, C., Piehler, J., Weninger, K., Riehn, R., Smith, S., Opersko, P. L., Wang, H.

Abstract:

The shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3' overhang (TPP1-POT1). Since TIN2 does not bind to DNA, determining its mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence-dependent manner. Pre-incubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 DNA bridges upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, and provides new insight into sister telomere cohesion.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.13.295436v1?rss=1

Authors: Diekman, C. O., Wei, N.

Abstract:

Sudden cardiac arrest is a malfunction of the heart's electrical system, typically caused by ventricular arrhythmias, that can lead to sudden cardiac death (SCD) within minutes. Epidemiological studies have shown that SCD and ventricular arrhythmias are more likely to occur in the morning than in the evening, and laboratory studies indicate that these daily rhythms in adverse cardiovascular events are at least partially under the control of the endogenous circadian timekeeping system. However, the biophysical mechanisms linking molecular circadian clocks to cardiac arrhythmogenesis are not fully understood. Recent experiments have shown that L-type calcium channels exhibit circadian rhythms in both expression and function in guinea pig ventricular cardiomyocytes. We developed an electrophysiological model of these cells to simulate the effect of circadian variation in L-type calcium conductance. We found that there is a circadian pattern in the occurrence of early afterdepolarizations (EADs), which are abnormal depolarizations during the repolarization phase of a cardiac action potential that can trigger fatal ventricular arrhythmias. Specifically, the model produces EADs in the morning but not at other times of day. We show that the model exhibits a codimension-2 Takens-Bogdanov bifurcation that serves as an organizing center for different types of EAD dynamics. We also simulated a 2-D spatial version of this model across a circadian cycle. We found that there is a circadian pattern in the breakup of spiral waves, which represents ventricular fibrillation in cardiac tissue. Specifically, the model produces spiral wave breakup in the morning but not in the evening. Our study is the first to propose a link between circadian rhythms and EAD formation and suggests that the efficacy of drugs targeting EAD-mediated arrhythmias may depend on the time of day that they are administered.

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