PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.27.315929v1?rss=1

Authors: Al-Shaer, A., Lyons, A., Ishikawa, Y., Hudson, B. G., Boudko, S., Forde, N. R.

Abstract:

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagens. In particular, network-forming collagen IV, an integral component of basement membranes, has been far less studied than fibril-forming collagens. A key feature differentiating collagen IV is the presence of interruptions in the triple-helix-defining (Gly-X-Y) sequence along its collagenous domain. Here, we used atomic force microscopy (AFM) to determine the impact of these interruptions on the flexibility of collagen. Our extracted flexibility profile reveals that collagen IV possesses highly heterogeneous mechanics, ranging from semi-flexible regions as found for fibril-forming collagens to a lengthy region of high flexibility towards its N terminus. A simple model in which flexibility is dictated only by the presence of interruptions fit the extracted profile reasonably well, providing insight into the alignment of chains and supporting the role of interruptions in instilling flexibility. However, limitations of this model were illuminated by our determination of variable flexibility along continuously triple-helical collagen III, which we found to possess a high-flexibility region around its matrix-metalloprotease (MMP) binding site. Surprisingly, proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are not used to control its compaction during secretion. Although extracellular chloride ions play a role in triggering collagen IV network formation, they do not appear to modulate the structure of its collagenous domain.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.27.315978v1?rss=1

Authors: Walshe, J. L., Patel, K., Ataide, S. F.

Abstract:

In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in dissociation from the DNA template. ANTAR domains are a class of antiterminator that bind and stabilise mutually exclusive dual hexaloop motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from E. faecialis, both in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate the conformational changes that occur upon RNA binding and reveal the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.27.316109v1?rss=1

Authors: Martynowycz, M. W., Shiriaeva, A., Ge, X., Hattne, J., Nannenga, B. L., Cherezov, V., Gonen, T.

Abstract:

G Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A2A receptor to 2.8 A resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.27.315614v1?rss=1

Authors: Wang, L., Noordanus, E., van Opstal, J. A.

Abstract:

The latency of the auditory steady-state response (ASSR) may provide valuable information regarding the integrity of the auditory system, as it could potentially reveal the presence of multiple intracerebral sources. To estimate multiple latencies from recorded ASSRs, we propose a novel two-stage procedure that consists of a nonparametric estimation method, called apparent latency from phase coherence (ALPC), followed by a heuristic sequential forward selection algorithm (SFS). Compared with existing methods, ALPC requires few prior assumptions, and is straightforward to implement for higher-order nonlinear responses to multi-cosine sound complexes with their initial phases set to zero. It systematically evaluates the nonlinear components of the ASSRs by estimating multiple latencies, automatically identifies involved ASSR components, and reports a latency consistency index (LCI). To verify the proposed method, we performed simulations for several scenarios: two nonlinear subsystems with different or overlapping outputs. We also recorded the EEG from ten normal-hearing adults by bilaterally presenting superimposed tones with four frequencies that evoke a unique set of ASSRs. From these ASSRs, two major latencies were found to be stable across subjects on repeated measurement days. The two latencies are dominated by low-frequency (LF) (near 40 Hz, at around 41-52 ms) and high-frequency (HF) (>80 Hz, at around 21-27 ms) ASSR components. The frontal-central (FC) brain region showed longer latencies on LF components, but shorter latencies on HF components, when compared with temporal-lobe regions. In conclusion, the proposed nonparametric ALPC-SFS method, applied to zero-phase, multi-cosine sound complexes is more suitable for evaluating embedded nonlinear systems underlying ASSRs than existing methods. It is thus promising as an objective measure of hearing performance and auditory cortex (dis)function.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.27.315606v1?rss=1

Authors: Cortese, D., Wan, K. Y.

Abstract:

Helical swimming is a ubiquitous strategy for motile cells to generate self-gradients for environmental sensing. The model biflagellate Chlamydomonas reinhardtii rotates at a constant 1 - 2 Hz as it swims, but the mechanism is unclear. Here, we show unequivocally that the rolling motion derives from a persistent, non-planar flagellar beat pattern. This is revealed by high-speed imaging and micromanipulation of live cells. We construct a fully-3D model to relate flagellar beating directly to the free-swimming trajectories. For realistic geometries, the model reproduces both the sense and magnitude of the axial rotation of live cells. We show that helical swimming requires further symmetry-breaking between the two flagella. These functional differences underlie all tactic responses, particularly phototaxis. We propose a control strategy by which cells steer towards or away from light by modulating the sign of biflagellar dominance.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.23.310425v1?rss=1

Authors: Auerswald, J., Ebenhan, J., Schwieger, C., Scrima, A., Meister, A., Bacia, K.

Abstract:

The insertion of protein domains into membranes is an important step in many membrane remodeling processes, for example in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. Total area changes in lipid monolayers can be measured on a Langmuir film balance. Finding the area per inserted protein however proves challenging for two reasons: The number of inserted proteins must be determined without disturbing the binding equilibrium and the change in the film area can be very small. Here we address both issues using Fluorescence Correlation Spectroscopy (FCS): Firstly, by labeling a fraction of the protein molecules fluorescently and performing FCS experiments directly on the monolayer, the number of inserted proteins is determined in situ without having to rely on invasive techniques, such as collecting the monolayer by aspiration. Secondly, by using another FCS color channel and adding a small fraction of fluorescent lipids, the reduction in fluorescent lipid density accompanying protein insertion can be monitored to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation, in a lipid monolayer. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 {+/-} 0.8) nm2 was obtained for Sar1 in monolayers from a lipid mixture typically used in reconstitution, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to be applied also to laterally heterogeneous monolayers and bilayers.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.25.313973v1?rss=1

Authors: Li, B., Ponjavic, A., Chen, W.-H., Hopkins, L., Hughes, C., Ye, Y., Bryant, C. E., Klenerman, D.

Abstract:

Detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2m thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-{beta}, which causes proteotoxicity relevant to Alzheimer's disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.25.313023v1?rss=1

Authors: Landajuela, A., Braun, M., Rodrigues, C. D. A., Doan, T., Horenkamp, F. A., Andronicos, A., Shteyn, V., Williams, N. D., Lin, C., Rudner, D. Z., Karatekin, E.

Abstract:

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, FisB, is expressed during sporulation in Bacillus subtilis and is required at the last stage of engulfment to release the developing spore into the mother cell cytoplasm. Here we characterize the requirements for FisB-mediated fission. FisB forms mobile clusters of ~12 molecules that give way to an immobile cluster at the engulfment pole containing ~40 proteins at the time of membrane fission. Reducing FisB to ~6 copies at the pole slowed but did not eliminate fission. Function mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Finally, a distant FisB homolog from Clostridium perfringens was functional in B. subtilis suggesting FisB catalyzes fission without protein partners. Our results suggest that FisB is a robust and unusual membrane fission protein that likely relies on homo-oligomerization, lipid-binding and the unique membrane topology generated during engulfment for localization and membrane scission.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.26.314609v1?rss=1

Authors: McBride, J. M., Tlusty, T.

Abstract:

Proteins are translated from the N- to the C-terminal, raising the basic question of how this innate directionality affects their evolution. To explore this question, we analyze 16,200 structures from the protein data bank (PDB). We find remarkable enrichment of -helices at the C terminal and {beta}-sheets at the N terminal. Furthermore, this -{beta} asymmetry correlates with sequence length and contact order, both determinants of folding rate, hinting at possible links to co-translational folding (CTF). Hence, we propose the slowest-first scheme, whereby protein sequences evolved structural asymmetry to accelerate CTF: the slowest-folding elements (e.g. {beta}-sheets) are positioned near the N terminal so they have more time to fold during translation. Our model predicts that CTF can be accelerated, up to double the rate, when folding time is commensurate with translation time; analysis of the PDB reveals that structural asymmetry is indeed maximal in this regime. This correspondence is greater in prokaryotes, which generally require faster protein production. Altogether, this indicates that accelerating CTF is a substantial evolutionary force whose interplay with stability and functionality is encoded in sequence asymmetry.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.26.315077v1?rss=1

Authors: Seifi, B., Wallin, S.

Abstract:

We study the folding and fold switching of the C-terminal domain (CTD) of the transcription factor RfaH using a hybrid sequence-structure based model. We show that this model captures the essential thermodynamic behavior of this metamorphic domain, i.e., a switch in the global free energy minimum from an -helical hairpin to a 5-stranded {beta}-barrel upon separating the CTD from the rest of the protein. Using this model and Monte Carlo sampling techniques, we analyze the energy landscape of the CTD in terms of progress variables for folding towards the two folds. We find that, below the folding temperature, the energy landscape is characterized by a single, dominant funnel to the native {beta}-barrel structure. The absence of a deep funnel to the -helical hairpin state reflects a negligible population of this fold for the isolated CTD. We observe, however, a significantly higher -helix structure content in the unfolded state compared to results from a similar but fold switch-incompetent version of our model. Moreover, in folding simulations started from an extended chain conformation we find transient -helix structure that disappears as the chain progresses to the thermally stable {beta}-barrel state.

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