PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.06.325969v1?rss=1

Authors: Tamura-Sakaguchi, R., Aruga, R., Hirose, M., Ekimoto, T., Miyake, T., Hizukuri, Y., Oi, R., Kaneko, M. K., Kato, Y., Akiyama, Y., Ikeguchi, M., Iwasaki, K., Nogi, T.

Abstract:

Antibody labeling has been extensively conducted for structure determination in both x-ray crystallography and EM analysis. However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, we have developed an alternative method to prepare an antibody-complex by inserting an exogenous epitope into the target. We here demonstrated that the Fab of monoclonal antibody NZ-1 could form a stable complex with the target containing an inserted PA14 tag as an epitope. By adopting a closed ring-like conformation inside the antigen-binding pocket, the inserted PA14 tag had less impact on the folding of the target. In fact, the PA14 tag could also be inserted into the sterically hindered loop for labeling. Using the improved labeling technique, we performed negative-stain EM on a bacterial site-2 protease, which enabled us to approximate the domain arrangement based on the docking mode of the NZ-1 Fab.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.05.326686v1?rss=1

Authors: Mitra, A., Choubey, S. K., Das, R., Rout, P., T.S., S., Mishra, D., Mandal, A.

Abstract:

Histopathology, the gold-standard method for diagnosis of human prostate cancer, is based on the analysis of changes in cellular morphology and tissue architecture in chemically stained tissue sections. Even at the very early stages of cancer with minimal phenotypic changes in cellular morphologies, there might be detectable changes in the expression profiles of proteins. Over the last decade, imaging mass spectrometry has been used to explore the spatial distribution and expression profiles of several molecules with their two-dimensional heterogeneity retained across the tissue section. In the present study, using MALDI mass spectrometry based tissue imaging, we report the differential expression of three proteins, vinculin, ribonuclease T2 and 60 kDa heat shock protein across human prostate tissue sections in regions pathologically demarcated as frankly malignant. In an independent analysis, quantitative proteomics revealed that in cancerous prostate tissues, ribonuclease T2 and 60 kDa heat shock protein were significantly overexpressed by 22.26- and 6 folds respectively compared to benign condition. Our results show the utility of this approach to probe differential protein expression in architecturally intact cancer tissues. In addition, we propose that ribonuclease T2 and 60 kDa heat shock protein might be developed as diagnostic biomarkers for prostate cancer in future.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.05.326595v1?rss=1

Authors: Pudasaini, A., Green, R., Song, Y. H., Blumenfeld, A., Karki, N., Imaizumi, T., Zoltowski, B. D.

Abstract:

Plants measure light, quality, intensity, and duration to coordinate growth and development with daily and seasonal changes in environmental conditions, however, the molecular details linking photochemistry to signal transduction remain incomplete. Two closely related Light, Oxygen, or Voltage (LOV) domain containing photoreceptor proteins ZEITLUPE (ZTL) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) divergently regulate the protein stability of circadian clock and photoperiodic flowering components to mediate daily and seasonal development. Using structural approaches, we identified that mutations at the Gly46 position led to global rearrangements of the ZTL dimer interface. Specifically, introduction of G46S and G46A variants that mimic equivalent residues found in FKF1 induce a 180{degrees} rotation about the dimer interface that is coupled to ordering of N- and C-terminal signaling elements. These conformational changes hinge upon rotation of a C-terminal Gln residue analogous to that present in light-state structures of ZTL. The results presented herein, confirm a divergent signaling mechanism within ZTL that deviates from other members of the LOV superfamily and suggests that mechanisms of signal transduction in LOV proteins may be fluid across the LOV protein family.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.05.327460v1?rss=1

Authors: Kang, A. S. W., Bernasconi, J. G., Jack, W., Kanavarioti, A.

Abstract:

Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies (ONT), and repurposed it to detect any DNA/RNA oligo (target) in a complex mixture by conducting voltage-driven ion-channel measurements. The detection and quantitation of the target is enabled by the use of a unique complementary probe. Using a validated labeling technology, probes are tagged with a bulky Osmium tag (Osmium tetroxide 2,2-bipyridine), in a way that preserves strong hybridization between probe and target. Intact oligos traverse the nanopore of the MinION relatively quickly compared to the acquisition rate of the device, and exhibit count of events comparable to the baseline. Counts are reported by a publicly available software, OsBp_detect. Due to the presence of the bulky Osmium tag, probes traverse more slowly, produce multiple counts over the baseline, and are even detected at single digit attomole (amole) range. In the presence of the target the probe is silenced. Silencing is attributed to a 1:1 double stranded (ds) complex that does not fit and can not traverse this nanopore. This ready-to-use platform can be tailored as a diagnostic test to meet the requirements for point-of-care cell-free tumor DNA (ctDNA) and microRNA (miRNA) detection and quantitation in body fluids.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.06.328401v1?rss=1

Authors: Jackson, V., Hermann, J., Tynan, C. J., Rolfe, D. J., Kalli, A. C., Duncan, A. L., Jones, E. Y., Sansom, M., Martin-Fernandez, M. L., Seiradake, E., Chavent, M.

Abstract:

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate synapse development and cell guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact in cis is remains unknown. Here, we reveal two dimerization motifs in the FLRT2 transmembrane helix. Molecular dynamics simulations and single particle tracking experiments show that these 'Small-X3-Small' motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and diffusion patterns of FLRT2 on cells. The results point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.06.327965v1?rss=1

Authors: Mazumder, A., Wang, A., Uhm, H., Ebright, R. H., Kapanidis, A. N.

Abstract:

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Forster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ~ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.06.327627v1?rss=1

Authors: Liu, Y., Elnatan, D., Sun, M., Myasnikov, A., Agard, D.

Abstract:

TRAP1 is a mitochondrion specific Hsp90, a ubiquitous chaperone family that mediates the folding and maturation of hundreds of 'client' proteins. Through the interaction with client proteins, TRAP1 regulates mitochondrial protein homeostasis, oxidative phosphorylation/glycolysis balance, and plays a critical role in mitochondrial dynamics and disease. However, the molecular mechanism of client protein recognition and remodeling by TRAP1 remains elusive. Here we established the succinate dehydrogenase B subunit (SdhB) from mitochondrial complex II as a client protein for TRAP1 amenable to detailed biochemical and structural investigation. SdhB accelerates the rate of TRAP1 dimer closure and ATP hydrolysis by 5-fold. Cryo-EM structures of the TRAP1:SdhB complex show TRAP1 stabilizes SdhB folding intermediates by trapping an SdhB segment in the TRAP1 lumen. Unexpectedly, client protein binding induces an asymmetric to symmetric transition in the TRAP1 closed state. Our results highlight a client binding mechanism conserved throughout Hsp90s that transcends the need for cochaperones and provide molecular insights into how TRAP1 modulates protein folding within mitochondria. Our structures also suggest a potential role for TRAP1 in Fe-S cluster biogenesis and mitochondrial protein import and will guide small molecule development for therapeutic intervention in specific TRAP1 client interactions.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.05.326330v1?rss=1

Authors: Ciorita, A., Bugiel, M., Sudhakar, S., Schaeffer, E., Jannasch, A.

Abstract:

Microtubules are highly dynamic cellular filaments and many intracellular processes like cell division depend on an accurate control of their length. Among other factors, microtubule length is actively modulated by motors from the kinesin superfamily. For example, yeast kinesin-8, Kip3, depolymerizes microtubules in a collective manner by a force and length-dependent mechanism. However, whether single motors depolymerize or stabilize microtubule ends is unclear. Here, using interference reflection microscopy, we measured the influence of single kinesin motors on the stability of microtubules in an in vitro assay. Surprisingly, using unlabeled, stabilized microtubules, we found that both single kinesin-8 and non-depolymerizing kinesin-1 transport motors stabilized microtubule ends further by reducing the spontaneous microtubule depolymerization rate. Since we observed this effect for two very different kinesins, it implies a more general stabilization mechanism. For Kip3, this behavior is contrary to the collective force-dependent depolymerization activity of multiple motors. The complex, concentration-dependent interaction with microtubule ends provides new insights into the molecular mechanism of kinesin-8 and its regulatory function of microtubule length.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.02.324442v1?rss=1

Authors: Patel, D., Patel, J. S., Ytreberg, F. M.

Abstract:

Protein-protein binding is fundamental to most biological processes. It is important to be able to use computation to accurately estimate the change in protein-protein binding free energy due to mutations in order to answer biological questions that would be experimentally challenging, laborious or time consuming. Although non-rigorous free energy methods are faster, rigorous alchemical molecular dynamics-based methods are considerably more accurate and are becoming more feasible with the advancement of computer hardware and molecular simulation software. Even with sufficient computational resources, there are still major challenges to using alchemical free energy methods for protein-protein complexes, such as generating hybrid structures and topologies, maintaining a neutral net charge of the system when there is a charge-changing mutation, and setting up the simulation. In the current study, we have used the pmx package to generate hybrid structures and topologies, and a double-system/single-box approach to maintain the net charge of the system. To test the approach, we predicted relative binding affinities for two protein-protein complexes using a non-equilibrium alchemical method based on the Crooks fluctuation theorem and compared the results with experimental values. The method correctly identified stabilizing from destabilizing mutations for a small protein-protein complex, but was not as successful to the larger, more challenging antibody complex. In addition, the correlation between predicted and experimental relative binding affinities was high for smaller complex, and low for the other larger complex.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.10.01.320507v1?rss=1

Authors: Paquette, M., Eichner, C., Knösche, T. R., Anwander, A.

Abstract:

The feasibility of non-invasive axonal diameter quantification with diffusion MRI is a strongly debated topic due to the neuroscientific potential of such information and its relevance for the axonal signal transmission speed. It has been shown that under ideal conditions, the minimal diameter producing detectable signal decay is bigger than most human axons in the brain, even using the strongest currently available MRI systems. We show that resolving the simplest situations including multiple diameters is unfeasible even with diameters much bigger than the diameter limit. Additionally, the recently proposed effective diameter resulting from fitting a single value over a distribution is almost exclusively influenced by the biggest axons. We show how impractical this metric is for comparing different distributions. Overall, axon diameters cannot be quantified by diffusion MRI in any relevant way.

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