PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.20.305581v1?rss=1

Authors: Woods, D. C., Rodríguez-Ropero, F., Wereszczynski, J.

Abstract:

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.20.305425v1?rss=1

Authors: Fernandes, D. D., Neale, C., Gomes, G.-N. W., Li, Y., Malik, A., Pandey, A., Orazietti, A., Wang, X., Ye, L., Prosser, S., Gradinaru, C. C.

Abstract:

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Forster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes [≥] 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 s) and slow (200-300 s) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.20.305243v1?rss=1

Authors: Gennerich, A., Liu, X., Rao, L.

Abstract:

Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.20.305060v1?rss=1

Authors: Ugarte La Torre, D. R., Takada, S. R.

Abstract:

Biological membranes have been prominent targets for coarse-grained (CG) molecular dynamics (MD) simulations. While minimal CG lipid models with three-beads per lipid and quantitative CG lipid models with >10-beads per lipid have been well studied, in between them, CG lipid models with a compatible resolution to residue-level CG protein models are much less developed. Here, we extended a previously developed three-bead lipid model into a five-bead model and parametrized it for two phospholipids, POPC and DPPC. The developed model, iSoLF, reproduced the area per lipid, hydrophobic thickness, and phase behaviors of the target phospholipid bilayer membranes at the physiological temperature. The model POPC and DPPC membranes were in liquid and gel phases, respectively, in accordance with experiments. We further examined the spontaneous formation of a membrane bilayer, the temperature dependence of physical properties, vesicle dynamics, and the POPC/DPPC two-component membrane dynamics of the CG lipid model, showing some promise. Once combined with standard C protein models, the iSoLF model will be a powerful tool to simulate large biological membrane systems made of lipids and proteins.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.20.305227v1?rss=1

Authors: Leijnse, N., Barooji, Y. F., Verhagen, B., Wullkopf, L., Erler, J., Semsey, S., Nylandsted, J., Doostmohammadi, A., Oddershede, L. B., Bendix, P. M. M.

Abstract:

Filopodia are actin-rich structures, present on the surface of practically every known eukaryotic cell. These structures play a pivotal role in specific cell-cell and cell-matrix interactions by allowing cells to explore their environment, generate mechanical forces, perform chemical signaling, or convey signals via intercellular tunneling nano-bridges. The dynamics of filopodia appear quite complex as they exhibit a rich behavior of buckling, pulling, length and shape changes. Here, we find that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling of their actin rich core. Importantly, we show the rotational dynamics of the filamentous actin inside filopodia for a range of highly distinct and cognate cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist, and hence rotation, is an emergent phenomenon of active filaments confined in a narrow channel which points to a generic mechanism present in all cells. Our measurements confirm that filopodia exert traction forces and form helical buckles in a range of different cell types that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.20.305359v1?rss=1

Authors: William, N. D., Landajuela, A., Kasula, R. K., Zhou, W., Powell, J. T., Xi, Z., Isaacs, F. J., Berro, J., Toomre, D., Karatekin, E., Lin, C.

Abstract:

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in mammalian and bacterial cells, tagged with fluorescent proteins or organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.18.302638v1?rss=1

Authors: Bonilla, S., Sherlock, M., MacFadden, A., Kieft, J. S.

Abstract:

Structured RNA elements are essential for biology and are ubiquitously used by viruses to control diverse infection-critical processes. Single-stranded RNA viruses often encode multiple processes into a single RNA element to maximize the functional capacity of their compact genomes. These important and elegant 'multifunctional' RNAs are hypothesized to use programmed structural changes to coordinate several, sometimes opposing, functions. However, detailed molecular mechanisms of such elements are largely mysterious because of the difficulty of solving dynamic RNA structures. We exploited recent advances in cryo-EM to directly visualize the architecture and infer conformational dynamics of the brome mosaic virus tRNA-like structure (BMV TLS), a multifunctional tRNA mimic that participates in replication, translation, and encapsidation of viral RNAs and that has eluded structure determination for decades. We found that although BMV TLS is aminoacylated by cellular tyrosyl-synthetase (TyrRS), its primary conformation is incompatible with TyrRS binding. Rather, the RNA is preorganized for replication, positioning the replicase promoter and the initiation site in close proximity to each other. An alternative 'tyrosylation-ready' conformation requires repositioning of a conformationally dynamic structural domain and this change must induce additional rearrangements that disrupt the 'replication-ready' configuration. These results demonstrate how programmed RNA dynamics can evolve to coordinate interactions with diverse cellular and viral proteins and thus organize multiple functions on a single RNA platform. Our results support the paradigm that RNA structures are inherently dynamic conformational ensembles, which enables multifunctionality. This work also highlights the emerging power of cryo-EM to dissect the dynamic conformational landscape of small discrete functional RNAs. Furthermore, we anticipate our method of rapidly mapping RNA domains within cryo-EM maps to be broadly useful.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.17.302299v1?rss=1

Authors: Quail, T., Golfier, S., Elsner, M., Ishihara, K., Julicher, F., Brugues, J.

Abstract:

Capillary forces are driven by interactions between liquids and surfaces. These forces are crucial for many processes ranging from biology and physics to engineering, such as the motion of aquatic insects on the surface of water, modulation of the material properties of spider silk, and self-assembly of small objects and microstructures. Recent studies have shown that cells assemble biomolecular condensates in a manner similar to phase separation. In the nucleus, these condensates are thought to drive transcription, heterochromatin formation, nucleolus assembly, and DNA repair. Here, we test if the interaction between liquid-like condensates and chromatin generates capillary forces, which might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, and theory to show that the pioneer transcription factor FoxA1 mediates the condensation of a DNA-protein phase via a mesoscopic first-order phase transition. Surprisingly, after nucleation, capillary forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA in close proximity using capillary forces. Our work suggests that the physics of DNA and protein condensation driven by capillary forces provides a general regulatory principle for chromatin organization.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.18.298562v1?rss=1

Authors: Peck, A., Yao, Q., Brewster, A. S., Zwart, P. H., Heumann, J. M., Sauter, N. K., Jensen, G. J.

Abstract:

Structure determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure but is constrained by the need for large, well-ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction, and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular weight threshold required by single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure determination methods. Here we present a data processing scheme that combines routines from X-ray crystallography and new algorithms we developed to solve structures from tomograms of nanocrystals. This pipeline handles image processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. We also assess the tolerance of this workflow to the effects of radiation damage. Our simulations indicate a trade-off between a wider tilt-range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors but not merging errors can be overcome with additional datasets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.18.304063v1?rss=1

Authors: Immadisetty, K., Sun, B., Kekenes-Huskey, P.

Abstract:

Parvalbumin (PV) is a calcium binding protein expressed in humans, fish and avian species. In these organisms, the calcium (Ca2+) affinities of specific PV isoforms can vary by orders of magnitude. Despite the availability of high resolution structural data for many PV isoforms, the structural bases for how such proteins confer widely-varying divalent Ca2+ affinities and selectivities against common ions like magnesium (Mg2+) has been difficult to rationalize. We therefore conducted molecular simulations of several -pavalbumin (-parvalbumin (PV)) constructs with Ca2+ affinities in the micromolar to nanomolar ranges to identify properties of conformations that contribute to their wide-ranging binding constants and selectivities against Mg2+. Specifically we examined D94S/G98E construct with a reported lower Ca2+ affinity ({approx} -18.2 kcal/mol) relative to the WT ({approx}-22 kcal/mol), an S55D/E59D variant with enhanced affinity ({approx}-24 kcal/mol), and a truncated variant of PV with weak affinity ({approx}-12.6 kcal/mol). We performed molecular dynamics simulations of these constructs and assessed their Ca2+ and Mg2+ binding properties using scores from molecular mechanics generalized Born approximation (MM/GBSA), ion/oxygen coordination patterns and thermodynamics via mean spherical approximation (MSA) theory, as well as via metrics of protein structure and hydration. Our key findings are that although MM/GBSA and MSA scores successfully rank-ordered the variants according to their previously-published affinities and Mg2+ selectivity, importantly, properties of Ca2+ loops in CBPs such as coordination, and charge are alone insufficient to rationalize their binding properties. Rather, Ca2+ affinity and selectivity against 2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions from protein folding and solubility. Our findings broaden our understanding of the molecular bases governing PV ion binding that are likely shared by many Ca2+ binding proteins.

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