PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.29.319400v1?rss=1

Authors: Hurley, M. E., Sheard, T. M. D., Norman, R., Kirton, H. M., Shah, S. S., Pervolaraki, E., Yang, Z., Gamper, N., White, E., Steele, D., Jayasinghe, I.

Abstract:

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.29.319475v1?rss=1

Authors: SINGH, R. K., Jonely, M., Leslie, E., Rejali, N. A., Noriega, R., Bass, B. L.

Abstract:

Drosophila melanogaster Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3{per thousand}-overhanging termini. We investigated the transient kinetic mechanism of these reactions using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. We found that ATP binding to dmDcr-2{per thousand}s helicase domain impacts the kinetics of dsRNA binding and dissociation in a termini-dependent manner, emphasizing the termini-dependent discrimination of dsRNA on a biologically-relevant time-scale. ATP-hydrolysis mediates local unwinding of dsRNA, and directional translocation on unwound single-stranded RNA, which is concurrent with a slow rewinding prior to dsRNA cleavage. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent change in conformational dynamics of the helicase and Platform.PAZ domains in the nanosecond timescale that is correlated with termini-dependent dsRNA cleavage. Our study delineates kinetic events and transient intermediates for a Dicer-catalyzed reaction, thus establishing a framework for understanding other Dicers and how accessory factors modulate the reaction.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.30.320069v1?rss=1

Authors: Gonzalez, T. R., Martin, K. P., Barnes, J. E., Patel, J. S., Ytreberg, F. M.

Abstract:

A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-antigen complexes for their ability to accurately predict relative binding affinities ({Delta}{Delta}G) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing ({Delta}{Delta}G < -0.5 kcal/mol) with high (83-98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-antigen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.30.320408v1?rss=1

Authors: Sobti, M., Walshe, J. L., Zeng, Y. C., Ishmukhametov, R., Stewart, A. G.

Abstract:

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F11 motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures of E. coli ATP synthase in different rotational and inhibited states observed following a 45 second incubation with 10 mM MgATP. The structures generated describe multiple changes that occur following addition of MgATP, with the inhibitory C-terminal domain of subunit {epsilon} ({epsilon}CTD) disassociating from the central stalk to adopt a condensed ''down'' conformation. The transition to the {epsilon}CTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by ~50{degrees}, with further flexing in the peripheral stalk enabling the c-ring to rotate by two sub-steps in the Fo motor. Truncation mutants lacking the second helix of the {epsilon}CTD suggest that central stalk rotational flexibility is important for F1Fo synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the {epsilon} subunit.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.29.319020v1?rss=1

Authors: Ripp, J., Kehrer, J., Smyrnakou, X., Tisch, N., Ruiz de Almodovar, C., Frischknecht, F.

Abstract:

Transmission of malaria-causing parasites to and by the mosquito rely on active parasite migration and constitute bottlenecks in the Plasmodium life cycle. Parasite adaption to the biochemically and physically different environments must hence be a key evolutionary driver for transmission efficiency. To probe how subtle but physiologically relevant changes in environmental elasticity impact parasite migration, we introduce 2D and 3D polyacrylamide gels to study ookinetes, the parasite forms emigrating from the mosquito blood meal and sporozoites, the forms transmitted to the vertebrate host. We show that ookinetes adapt their migratory path but not their speed to environmental elasticity and are motile for over 24 hours on soft substrates. In contrast, sporozoites evolved more short-lived rapid gliding motility for rapidly crossing the skin. Strikingly, sporozoites are highly sensitive to substrate elasticity possibly to avoid adhesion on soft endothelial cells on their long way to the liver. Hence the two migratory stages of Plasmodium evolved different strategies to overcome the physical challenges posed by the respective environments and barriers they encounter.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.30.320812v1?rss=1

Authors: Tack, D. S., Tonner, P. D., Pressman, A., Olson, N. D., Levy, S. F., Romantseva, E. F., Alperovich, N., Vasilyeva, O., Ross, D.

Abstract:

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 10^5 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.317057v1?rss=1

Authors: Sgouralis, I., Bryan, J. S., Presse, S.

Abstract:

Determining the number of biomolecules within a small, diffraction-limited spot, is key to understanding intermolecular interactions that form the basis of all of information processing relevant to life. To enumerate the number of biomolecules within such a small region of interest (ROI), it is possible to illuminate an ROI containing a collection of fluorescently labeled biomolecules and observe the ROI's brightness over time. As most fluorescent labels (fluorophores) are initially in a fluorescently active state, the brightness diminishes in a step-like pattern as fluorophores photobleach one at a time. Naively, by counting steps, one could determine the number of fluorophores present. However, this analysis is complicated by photon shot noise, camera noise that amplifies intrinsic photon shot noise and fluorophore photophysics (i.e., states with variable emission visited by fluorophores). Current state of the art can typically enumerate as many as 20 fluorophores reliably. Yet in many biophysics problems, fluorophore numbers can vastly exceed 20 within an ROI. For this reason, we develop a method to learn the number of fluorophores alongside other parameters required to achieve high counts simultaneously and self-consistently (e.g., fluorophore photophysical rate parameters and camera parameters). As the number of fluorophores is initially unknown and may be very large, and as not all fluorophores may initially be emitting photons, we cannot rely on a traditional (parametric) Bayesian framework. As such, to achieve our goal of exceeding the state of the art by a factor of about 5, we must abandon the parametric Bayesian paradigm and invoke novel tools within Bayesian nonparametrics never previously used in step counting by photobleaching.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.30.320762v1?rss=1

Authors: Krichel, B., Bylapudi, G., Schmidt, C., Blanchet, C., Schubert, R., Brings, L., Koehler, M., Zenobi, R., Svergun, D., Lorenzen, K., Madhugiri, R., Ziebuhr, J., Uetrecht, C.

Abstract:

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.316596v1?rss=1

Authors: Santra, M., Seal, A., Bhattacharjee, K., Chakrabarty, S.

Abstract:

Photoisomerisation in retinal leads to a channel opening in the rhodopsins that triggers translocation of an ion/proton. Crystal structures of rhodopsins contain several structurally conserved water molecules. It has been suggested that water plays an active role in facilitating the ion pumping/translocation process by acting as a lubricant in these systems. In this work, we investigate the localisation, local structure and dynamics of water molecules along the channel for the resting/dark state of KR2 rhodopsin. Employing 1.5 s long atomistic molecular dynamics (MD) simulations of this trans-membrane protein system, we demonstrate the presence of five distinct water containing pockets/cavities separated by gateways controlled by the protein side-chains. We present evidence of significant structural and dynamical heterogeneity in the water molecules present in these cavities. The exchange time-scale of these buried water with bulk ranges from tens of nanoseconds to >1.5 s. The translational and rotational dynamics of buried water are found to be strongly dependent on protein cavity size and local interactions with possible functional significance.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.28.316547v1?rss=1

Authors: Michieletto, D.

Abstract:

Systems of "living' polymers are ubiquitous in industry and are traditionally realised using surfactants. Here I review the state-of-the-art of living polymers and discuss non-equilibrium extensions that may be realised with advanced synthetic chemistry or DNA functionalised by proteins. These systems are not only interesting in order to realise novel "living" soft matter but can also shed insight into how genomes are (topologically) regulated in vivo.

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