PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.317487v1?rss=1

Authors: Llorca, O., Lopez-Perrote, A., Hug, N., Gonzalez-Corpas, A., Rodriguez, C. F., Serna, M., Garcia-Martin, C., Boskovic, J., Fernandez-Leiro, R., Caceres, J. F.

Abstract:

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.317339v1?rss=1

Authors: Orozco, C. T., Edgeworth, M. J., Devine, P. W. A., Hines, A. R., Cornwell, O., Wang, X., Phillips, J. J., Ravn, P., Jackson, S. E., Bond, N. J.

Abstract:

Antibody drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterise an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts and changes the stability and structural dynamics of the antibody. Insight is gained into the local and global destabilisation associated with the engineering and subsequent disulfide bonded variant that will inform future engineering strategies.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.315325v1?rss=1

Authors: Camino, J. D., Gracia, P., Chen, S. W., Sot, J., de la Arada, I., Sebastian, V., Arrondo, J. L. R., Goni, F., Dobson, C. M., Cremades, N.

Abstract:

-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson's disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel {beta}-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that -synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel {beta}-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside -synuclein droplets generated by liquid-liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo -synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.316729v1?rss=1

Authors: LAPLAUD, V., Levernier, N., Pineau, J., San-Roman, M., Barbier, L., Saez, P. J., Lennon-Dumenil, A.-M., Vargas, P., Kruse, K., DU ROURE, O., Piel, M., Heuvingh, J.

Abstract:

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration and division. Because the cortical thickness is below optical resolution, it has been generally considered as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension due to actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape changing capacity of highly contractile cells that use amoeboid-like migration.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.306787v1?rss=1

Authors: Roopchansingh, V., French, J. J., Nielson, D. M., Reynolds, R. C., Glen, D. R., D'Souza, P., Taylor, P. A., Cox, R. W., Thurm, A. E.

Abstract:

Task, resting state, and diffusion MRI data are usually acquired from subjects using echo-planar based imaging techniques. These techniques are highly susceptible to B0 homogeneity effects that result in geometric distortions in the reconstructed images. As researchers work to link the information from these scans back to various developmental stages, or to conditions and diseases in specific regions or structures of the brain, it becomes critical to have accurate correspondence between more geometrically distorted echo-planar images and less geometrically distorted anatomical images. A variety of techniques and tools have been developed to improve this correspondence. The basic premise behind most techniques used to mitigate geometric distortion is to acquire enough information to inform software tools how echo-planar images are warped, then have them undo that warping. Here, we investigate the application of two common methods: B0 correction, and reverse-polarity phase-encoding (or reverse blip) correction. We implement each of these in two separate, widely used software packages in the field: AFNI and FSL. We find that using either technique in either software package results in reduced geometric distortions in the EPI images. We discuss the practical implementations of these methods (e.g., increased scan and processing time). In general, however, both methods possess readily available data acquisition schemes, and are highly efficient to include in processing streams. Due to the overall data improvement, we strongly recommend that researchers include one of these methods in their standard protocols.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.316430v1?rss=1

Authors: Bauer, A., Prechova, M., Gregor, M., Fabry, B.

Abstract:

Cellular force generation and force transduction are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on single cells, cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/ ) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints . In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. The Monolayer Stress Microscopy implementation in pyTFM allows for the analysis of small cell patches and single cells; we analyze the accuracy and performance of Traction Force Microscopy and Monolayer Stress Microscopy algorithms using synthetic and experimental data from epithelial cell patches.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.317040v1?rss=1

Authors: Cagiada, M., Johansson, K. E., Valanciute, A., Nielsen, S. V., Hartmann-Petersen, R., Yang, J. J., Fowler, D. M., Stein, A., Lindorff-Larsen, K.

Abstract:

Understanding and predicting how amino acid substitutions affect proteins is key to practical uses of proteins, and to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analysed 6749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects, and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyse the structural and mechanistic origins of loss of function, and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.317206v1?rss=1

Authors: Ghorbani, M., Brooks, B. R., Klauda, J. B.

Abstract:

The ongoing pandemic caused by coronavirus SARS-COV-2 continues to rage with devastating consequences on human health and global economy. The spike glycoprotein on the surface of coronavirus mediates its entry into host cells and is the target of all current antibody design efforts to neutralize the virus. The glycan shield of the spike helps the virus to evade the human immune response by providing a thick sugar-coated barrier against any antibody. To study the dynamic motion of glycans in the spike protein, we performed microsecond-long MD simulation in two different states that correspond to the receptor binding domain in open or closed conformations. Analysis of this microsecond-long simulation revealed a scissoring motion on the N-terminal domain of neighboring monomers in the spike trimer. Role of multiple glycans in shielding of spike protein in different regions were uncovered by a network analysis, where the high betweenness centrality of glycans at the apex revealed their importance and function in the glycan shield. Microdomains of glycans were identified featuring a high degree of intra-communication in these microdomains. An antibody overlap analysis revealed the glycan microdomains as well as individual glycans that inhibit access to the antibody epitopes on the spike protein. Overall, the results of this study provide detailed understanding of the spike glycan shield, which may be utilized for therapeutic efforts against this crisis.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.316307v1?rss=1

Authors: Jin, F., Wang, Y., Wang, M., Sun, M., Hattori, M.

Abstract:

Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.27.315796v1?rss=1

Authors: Bauer, M. S., Gruber, S., Milles, L. F., Nicolaus, T., Schendel, L. C., Gaub, H. E., Lipfert, J.

Abstract:

The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within [≤]1 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

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