PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.20.347542v1?rss=1

Authors: Lichtinger, S. M., Garaizar, A., Collepardo-Guevara, R., Reinhardt, A.

Abstract:

Rationally and efficiently modifying the amino-acid sequence of proteins to control their ability to undergo liquid-liquid phase separation (LLPS) on demand is not only highly desirable, but can also help to elucidate which protein features are important for LLPS. Here, we propose an innovative computational method that couples a genetic algorithm to a sequence-dependent coarse-grained protein model to evolve the amino-acid sequences of phase-separating intrinsically disordered protein regions (IDRs), and purposely enhance or inhibit their capacity to phase-separate. We apply it to the phase-separating IDRs of three naturally occurring proteins, namely FUS, hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction. We find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids. However, the direction of change in the molecular driving forces that enhance LLPS (such as hydrophobicity, aromaticity and charge) depends on the initial amino-acid sequence: the critical temperature can be enhanced by increasing the frequency of hydrophobic and aromatic residues, by changing the charge patterning, or by a combination of both. Finally, we show that the evolution of amino-acid sequences to modulate LLPS is strongly coupled to the composition of the medium (e.g. the presence or absence of RNA), which may have significant implications for our understanding of phase separation within the many-component mixtures of biological systems.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.20.346973v1?rss=1

Authors: Haldar, S., chaudhuri, D., banerjee, s., Chakraborty, S.

Abstract:

Protein folding under force is an integral source of generating mechanical power to carry out diverse cellular functions. Though chaperones interact with proteins throughout the different stages of folding pathways, how they behave and interact with client proteins under force was not known. Here we introduce the mechanical role of chaperone and explained it with seven independent chaperones using single molecule based real-time microfluidics-magnetic-tweezers. We showed and quantified how chaperones increase or decrease mechanical work output by shifting the folding energy landscape of the client proteins towards the folded or unfolded state. Notably, we found chaperones could behave differently under force. For instance: trigger factor, ribosomal-tunnel associated chaperone, working as a holdase in absence of force, but assist folding under force. This phenomenon generates extra mechanical energy to pull the polyprotein from the stalled ribosome. This is also relevant for SecYEG tunnel associated oxidoreductase DsbA, which act similarly like TF and increases the mechanical energy up to ~59 zJ, to facilitate membrane translocation in an energy efficient manner. However cytoplasmic oxidoreductases such as PDI and Thioredoxin, unlike DsbA, do not have the mechanical folding ability. Interestingly, we observed a highly potential foldase- DnaKJE chaperone complex, only restores the folding ability of the client protein and fails to act like TF or DsbA under force. However, the individual components of this complex, DnaK or DnaJ, act as a mechanical holdase and inhibits folding; similar to that of SecB. Together our study provides an emerging insight of mechanical chaperone behavior, where tunnel associated chaperones generate extra mechanical work whereas the cytoplasmic chaperones are unable to generate that, which might have evolved to minimize the energy consumption in biological processes.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.20.346684v1?rss=1

Authors: Joiret, M., Rapino, F., Close, P., Geris, L.

Abstract:

The impact of the ribosome exit tunnel electrostatics on the protein elongation rate or on the forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in prokaryotes. Here, we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show how the tunnel geometry causes a positive potential difference between the tunnel exit and entry points which impedes positively charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of minus 40% to plus 85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derived, at single residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall and the shape and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, structural and physical chemistry, biomechanics, synthetic and multi-omics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control and into the role of the ribosome's mechanochemistry in the co-translational protein folding.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.20.346882v1?rss=1

Authors: Noa, A., Kuan, H.-S., Aschmann, V., Zaburdaev, V., Hilbert, L.

Abstract:

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such domain-within-domain packing, also called hierarchical packing, is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into a hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. In particular, we can pinpoint the values required to mimic a microphase-separated configuration of euchromatin. We suspect that the concept of multiplicative cascades can also be applied to images of other types of chromatin. In particular, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.21.348904v1?rss=1

Authors: Vala, M., Bujak, L., Marin, A. G., Holanova, K., Henrichs, V., Braun, M., Lansky, Z., Piliarik, M.

Abstract:

Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with enrichment of ordered protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a mechanism of switching from the disassembly to the assembly phase.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.20.347641v1?rss=1

Authors: Amitai, A.

Abstract:

The evolution of circulating viruses is shaped by their need to evade the adaptive immune system. The spike protein which mediates entry to the host cell is the main target of antibody response. Because of the dense presentation of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies, leading to complex immunodominance patterns. We used 3D coarse-grained computational models to estimate the antibody pressure on the seasonal flu H1N1 and the SARS subgenus spikes. Analyzing publically available sequences, we show that antibody pressure, through the geometrical organization of these spikes on the viral surface, shaped their mutability. Studying the mutability patterns of SARS-CoV-2 and the 2009 H1N1 pandemic spikes, we find that they are not predominantly shaped by antibody pressure. However, for SARS-CoV-2, we find that over time, it acquired, at low frequency, several mutations at antibody-accessible positions, which could indicate possible escape as define by our model. Hence, we offer a geometry-based approach to estimate and assess whether a pandemic virus is changing its mutational pattern to that indicative of a circulating virus.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.19.344994v1?rss=1

Authors: Narayana Iyengar, S. T., Kumar, T., Martensson, G., Russom, A.

Abstract:

Improved sample preparation has the potential to address a huge unmet need for fast turnaround sepsis tests that enable early administration of appropriate antimicrobial therapy. In recent years, inertial and elasto-inertial microfluidics-based sample preparation has gained substantial interest for bioparticle separation applications. However, for applications in blood stream infections the throughput and bacteria separation efficiency has thus far been limited. In this work, for the first time we report elasto-inertial microfluidics-based bacteria isolation from blood at throughputs and efficiencies unparalleled with current microfluidics-based state of the art. In the method, bacteria-spiked blood sample is prepositioned close to the outer wall of a spiral microchannel using a viscoelastic sheath buffer. The blood cells will remain fully focused throughout the length of the channel while bacteria migrate to the inner wall for effective separation. Initially, particles of different sizes were used to investigate particle focusing and the separation performance of the spiral device. A separation efficiency of 96% for the 1 micrometer particles was achieved, while 100% of 3 micrometer particles were recovered at the desired outlet at a high throughput of 1 mL/min. Following, processing blood samples revealed a minimum of 1:2 dilution was necessary to keep the blood cells fully focus at the outer wall. In experiments involving bacteria spiked in diluted blood, viable E.coli were continuously separated at a total flow rate of 1 mL/min, with an efficiency between 82 to 90% depending on the blood dilution. Using a single spiral, it takes 40 minutes to process 1 mL of blood at a separation efficiency of 82% and 3 hours at 90% efficiency. To the best of our knowledge, this is the highest blood sample throughput per single microfluidic chip reported for the corresponding separation efficiency. As such, the label-free, passive and high throughput bacteria isolation method has a great potential for speeding up downstream phenotypic and molecular analysis of bacteria.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.18.344648v1?rss=1

Authors: Kato, K., Miyazaki, N., Hamaguchi, T., Nakajima, Y., Akita, F., Yonekura, K., Shen, J.-R.

Abstract:

Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state, and its final state structure has not been solved because of the low efficiencies of the S-state transitions in the crystals. Here we analyzed the structure of PSII in solution at 1.95 [A] resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, which was reduced by reducing the electron dose. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.19.344408v1?rss=1

Authors: Kazantsev, A., Ignatova, Z.

Abstract:

G{whitebullet}U(T) mismatch has the highest contribution to the error rate of base pair recognition in replication, as well as in codon-anticodon decoding in translation. Recently, this effect was unambiguously linked to keto-enol tautomerization, which enables the Watson-Crick (WC) geometry of the base pair. Structural studies of the ribosome revealing G{whitebullet}U in the WC geometry in the closed state of the A-site challenge the canonical induced-fit model of decoding and currently lack a physicochemical explanation. Using computational and theoretical methods, we herein address effects of the ribosomal A-site on the wobble{leftrightarrow}WC tautomerization reaction in G{whitebullet}U (wb{leftrightarrow}WC reaction), and the consequent implications for the decoding mechanism in translation. The free energy change of the wb-WC reaction in the middle codon-anticodon position was calculated with quantum-mechanical/molecular-mechanical (QM/MM) umbrella sampling simulations. The wb-WC reaction was endoergic in the open A-site, but exoergic in the closed state. This effect can be explained in part by the decreased polarity of the closed A-site. We developed a model of initial selection in translation that incorporates the wb-WC reaction parameters in the open and closed states of the A-site. In the new model the exoergic wb-WC reaction is kinetically restricted by the decoding rates, which explains the observations of the WC geometry at equilibrium conditions. Moreover, the model reveals constraints imposed by the exoergic wb-WC reaction on the decoding accuracy: its equilibration counteracts the favorable contribution from equilibration of the open-closed transition. The similarity of the base-pair recognition mechanism in DNA polymerases allows extending this model to replication as well. Our model can be a next step towards a general recognition model for flexible substrates.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.19.346023v1?rss=1

Authors: Barros, E. P., Demir, O., Soto, J., Cocco, M. J., Amaro, R. E.

Abstract:

The tumor suppressor p53 is the most frequently mutated gene in human cancer, and thus reactivation of mutated p53 is a promising avenue for cancer therapy. Analysis of wildtype p53 and the Y220C cancer mutant long-timescale molecular dynamics simulations with Markov state models and validation by NMR relaxation studies has uncovered the involvement of loop L6 in the slowest motions of the protein. Due to its distant location from the DNA-binding surface, the conformational dynamics of this loop has so far remained largely unexplored. We observe mutation-induced stabilization of alternate L6 conformations, distinct from all experimentally-determined structures, in which the loop is both extended and located further away from the DNA-interacting surface. Additionally, the effect of the L6-adjacent Y220C mutation on the conformational landscape of the functionally-important loop L1 suggests an allosteric role to this dynamic loop and the inactivation mechanism of the mutation. Finally, the simulations reveal a novel Y220C cryptic pocket that can be targeted for p53 rescue efforts. Our approach exemplifies the power of the MSM methodology for uncovering intrinsic dynamic and kinetic differences among distinct protein ensembles, such as for the investigation of mutation effects on protein function.

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