Audio versions of bioRxiv paper abstracts
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.21.352724v1?rss=1 Authors: Anderies, B. J., Yee, S. F., Jackson, P. R., Rickertsen, C. R., Hawkins-Daarud, A. J., Johnston, S. K., Clark-Swanson, K. R., Hoxworth, J. M., Le, Y., Zhou, Y., Pepin, K. M., Massey, S. C., Hu, L. S., Huston, J. R., Swanson, K. R. Abstract: Gliomas are brain tumors characterized by highly variable growth patterns. Magnetic resonance imaging (MRI) is the cornerstone of glioma diagnosis and management planning. However, glioma features on MRI do not directly correlate with tumor cell distribution. Additionally, there is evidence that glioma tumor characteristics and prognosis are sex-dependent. Magnetic resonance elastography (MRE) is an imaging technique that allows interrogation of tissue stiffness in-vivo and has found utility in the imaging of several cancers. We investigate the relationship between MRI features, MRE features, and growth parameters derived from an established mathematical model of glioma proliferation and invasion. Results suggest that both the relationship between tumor volume and tumor stiffness as well as the relationship between the parameters derived from the mathematical model and tumor stiffness are sex-dependent. These findings lend evidence to a growing body of knowledge about the clinical importance of sex in the context of cancer diagnosis, prognosis and treatment. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.391862v1?rss=1 Authors: Ayoubi, M., van Tol, A. F., Weinkamer, R., Roschger, P., Brugger, P., Berzlanovich, A., Bertinetti, L., Roschger, A., Fratzl, P. Abstract: During bone remodeling, osteoblasts are known to deposit unmineralized collagenous tissue (osteoid), which mineralizes after some time lag. Some of the osteoblasts differentiate into osteocytes, forming a cell network within the lacunocanalicular network (LCN) of bone. To get more insight into the potential role of osteocytes in the mineralization process of osteoid, sites of bone formation were three-dimensionally imaged in nine forming human osteons using focused ion beam-scanning electron microscopy (FIB-SEM). In agreement with previous observations, the mineral concentration was found to gradually increase from the central Haversian canal towards preexisting mineralized bone. Most interestingly, a similar feature was discovered on a length scale more than 100-times smaller, whereby mineral concentration increased from the LCN, leaving around the canaliculi a zone virtually free of mineral, the size of which decreases with progressing mineralization. This suggests that the LCN controls mineral formation but not just by diffusion of mineralization precursors, which would lead to a continuous decrease of mineral concentration from the LCN. Our observation is, however, compatible with the co-diffusion and reaction of precursors and inhibitors from the LCN into the bone matrix. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.392225v1?rss=1 Authors: Yang, X., Needleman, D. J. Abstract: Mitochondria are central to metabolism and their dysfunctions are associated with many diseases. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo. However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria. Here we show that, if properly interpreted, FLIM of NADH can be used to quantitatively measure the flux through a major mitochondrial metabolic pathway, the electron transport chain (ETC), in vivo with subcellular resolution. This result is based on the use of a coarse-grained NADH redox model, which we test in mouse oocytes subject to a wide variety of perturbations by comparing predicted fluxes to direct biochemical measurements and by self-consistency criterion. Using this method, we discovered a subcellular spatial gradient of mitochondrial metabolic flux in mouse oocytes. We showed that this subcellular variation in mitochondrial flux correlates with a corresponding subcellular variation in mitochondrial membrane potential. The developed model, and the resulting procedure for analyzing FLIM of NADH, are valid under nearly all circumstances of biological interest. Thus, this approach is a general procedure to measure metabolic fluxes dynamically in living cells, with subcellular resolution. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390005v1?rss=1 Authors: van Krugten, J., Danne, N., Peterman, E. J. G. Abstract: Sensing and reacting to the environment is essential for survival and procreation of most organisms. Caenorhabditis elegans senses soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Development, maintenance and function of these cilia relies on intraflagellar transport (IFT), in which motor proteins transport cargo, including sensory TPs, back and forth along the ciliary axoneme. Here we use live fluorescence imaging to show that IFT machinery and the sensory TP OCR-2 reversibly redistribute along the cilium after exposure to repellant chemicals. To elucidate the underlying mechanisms, we performed single-molecule tracking experiments and found that OCR-2 distribution depends on an intricate interplay between IFT-driven transport, normal diffusion and subdiffusion that depends on the specific location in the cilium. These insights in the role of IFT on the dynamics of cellular signal transduction contribute to a deeper understanding of the regulation of sensory TPs and chemosensing. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390187v1?rss=1 Authors: Casalino, L., Dommer, A. C., Gaieb, Z., Barros, E. P., Sztain, T., Ahn, S.-H., Trifan, A., Brace, A., Ma, H., Lee, H., Turilli, M., Bogetti, A., Khalid, S., Chong, L., Simmerling, C., Hardy, D., Maia, J., Phillips, J., Kurth, T., Stern, A., Huang, L., McCalpain, J., Tatineni, M., Gibbs, T., Stone, J. E., Jha, S., Ramanathan, A., Amaro, R. E. Abstract: We develop a generalizable AI-driven workflow that leverages heterogeneous HPC resources to explore the time-dependent dynamics of molecular systems. We use this workflow to investigate the mechanisms of infectivity of the SARS-CoV-2 spike protein, the main viral infection machinery. Our workflow enables more efficient investigation of spike dynamics in a variety of complex environments, including within a complete SARS-CoV-2 viral envelope simulation, which contains 305 million atoms and shows strong scaling on ORNL Summit using NAMD. We present several novel scientific discoveries, including the elucidation of the spike's full glycan shield, the role of spike glycans in modulating the infectivity of the virus, and the characterization of the flexible interactions between the spike and the human ACE2 receptor. We also demonstrate how AI can accelerate conformational sampling across different systems and pave the way for the future application of such methods to additional studies in SARS-CoV-2 and other molecular systems. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.389106v1?rss=1 Authors: Jetta, D., Bahrani Fard, M. R., Sachs, F., Munechika, K., Hua, S. Z. Abstract: Adherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388744v1?rss=1 Authors: Pang, T. Y., Lercher, M. Abstract: A substantial fraction of the bacterial cytosol is occupied by catalysts and their substrates. While a higher volume density of catalysts and substrates might boost biochemical fluxes, the resulting molecular crowding can slow down diffusion, perturb the reactions' Gibbs free energies, and reduce the catalytic efficiency of proteins. Due to these tradeoffs, dry mass density likely possesses an optimum that facilitates maximal cellular growth and that is interdependent on the cytosolic molecule size distribution. Here, we analyse the balanced growth of a model cell with metabolic and ribosomal reactions, accounting systematically for crowding effects on reaction kinetics. We find that changes in cytosolic density affect biochemical efficiency more strongly for ribosomal reactions than for metabolic reactions, which involve much smaller catalysts and reactants. Accordingly, optimal cytosolic density depends on cellular resource allocation into ribosomal vs. metabolic reactions. A shift in the relative contributions of these sectors to the cellular economy explains the 10% difference in the cytosolic density between E. coli bacteria growing in nutrient-rich and -poor environments. We conclude that cytosolic density variation in E. coli is consistent with an optimality principle of cellular efficiency. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.388801v1?rss=1 Authors: Vahalova, P., Cervinkova, K., Cifra, M. Abstract: Nowadays, modern medicine is looking for new, more gentle and more efficient diagnostic methods. A pathological state of an organism is often closely connected with increased amount of reactive oxygen species (ROS). They can react with biomolecules and subsequent reactions can lead to very low endogenous light emission (biological autoluminescence - BAL). This phenomenon can be potentially used as a non-invasive and low-operation-cost tool for monitoring oxidative stress during diseases. To contribute to the understanding of the parameters affecting BAL, we analyzed the BAL from yeast Saccharomyces cerevisiae as a representative eukaryotic organism, The relationship between the BAL intensity and amount of ROS that originates as a result of the Fenton reaction as well as the correlation between spontaneous BAL and selected physical and chemical parameters (pH, oxygen concentration, and cell concentration) during cell growth were established. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.391409v1?rss=1 Authors: Tempra, C., La Rosa, C., Lolicato, F. Abstract: The most accredited hypothesis links the toxicity of amyloid proteins to their harmful effects on membrane integrity through the formation of prefibrillar-transient oligomers able to disrupt cell membranes. However, damage mechanisms necessarily assume a first step in which the amyloidogenic protein transfers from the aqueous phase to the membrane hydrophobic core. This determinant step is still poorly understood. However, according to our lipid-chaperon hypothesis, free lipids in solution play a crucial role in facilitating this footfall. Free phospholipid concentration in the aqueous phase acts as a switch between ion channel-like pore and fibril formation, so that high free lipid concentration in solution promotes pore and repress fibril formation. Conversely, low free lipids in the solution favor fibril and repress pore formation. This behavior is due to the formation of stable lipid-protein complexes. Here, we hypothesize that the helix propensity is a fundamental requirement to fulfill the lipid-chaperon model. The alpha-helix region seems to be responsible for the binding with amphiphilic molecules fostering the proposed mechanism. Indeed, our results show the dependency of protein-lipid binding from the helical structure presence. When the helix content is substantially lower than the wild type, the contact probability decreases. Instead, if the helix is broadening, the contact probability increases. Our findings open a new perspective for in silico screening of secondary structure-targeting drugs of amyloidogenic proteins. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.20.390328v1?rss=1 Authors: McFerrin, K. G., Pang, Y.-P. Abstract: Hemicarcerands are host molecules created to study constrictive binding with guest molecules for insights into the rules of molecular complexation. However, the molecular dynamics simulations that facilitate such studies have been limited because three-dimensional models of hemicarcerands are tedious to build and their atomic charges are complicated to derive. There have been no molecular dynamics simulations of the reported water-soluble hemicarcerand (Octacid4) that explain how it uniquely encapsulates its guests at 298 K and keeps them encapsulated at 298 K in NMR experiments. Herein we report a modular approach to hemicarcerand simulations that simplifies the model building and charge derivation in a manner reminiscent of the approach to protein simulations with truncated amino acids as building blocks. We also report that apo Octacid4 in water adopts two clusters of conformations, one of which has an equatorial portal open thus allowing guests to enter the cavity of Octacid4, in microsecond molecular dynamics simulations performed using the modular approach at 298 K. Under the same simulation conditions, the guest-bound Octacid4 adopts one cluster of conformations with all equatorial portals closed thus keeping the guests incarcerated. These results explain the unique constrictive binding of Octacid4 and suggest that the guest-induced host conformational change that impedes decomplexation is a previously unrecognized conformational characteristic that promotes strong molecular complexation. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388843v1?rss=1 Authors: Van Oort, C. M., Ferrell, J. B., Remington, J. M., Wshah, S., Li, J. Abstract: Antibiotic resistance is a critical public health problem. Each year ${sim}2.8$ million resistant infections lead to more than 35,000 deaths in the U.S. alone. Anti-microbial peptides (AMPs) show promise in treating resistant infections. But, applications of known AMPs have encountered issues in development, production, and shelf-life. To drive the development of AMP-based treatments it is necessary to create design approaches with higher precision and selectivity towards resistant targets. In this paper we present AMPGAN v2, a generative adversarial network (GAN) based approach for rational AMP design. Like AMPGAN, AMPGAN v2 combines data driven priors and controlled generation. These elements allow for the generation of AMP candidates tailored for specific applications. AMPGAN v2 is able to generate AMP candidates that are novel and diverse, making it an efficient AMP design tool. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388561v1?rss=1 Authors: Medina, L., Gonzalez Lizarraga, F., Dominguez Meijide, A., Ploper, D., Parrales, V., Sequeira, S., Cima Omorri, M. S., Zweckstetter, M., del Bel, E., Michel, P. P., Fleming Outeiro, T., Raisman-Vozari, R., Chehin, R. N., Socias, S. B. Abstract: Tauopathies are neurodegenerative disorders with increasing incidence and still without cure. The extensive time required for development and approval of novel therapeutics highlights the need for testing and repurposing known safe molecules. Since doxycycline impacts alpha-synuclein aggregation and toxicity, herein we tested its effect on tau. We found that doxycycline reduces amyloid aggregation of the different isoforms of tau protein in a dose-dependent manner, remodeling the resultant species. Furthermore, doxycycline interacts with tau microtubule-binding domain preventing its aggregation. In a cell free system doxycycline also prevents tau seeding and in cell culture reduces toxicity of tau aggregates. Overall, our results expand the spectrum of action of doxycycline against aggregation-prone proteins, opening novel perspectives for its repurposing as a disease-modifying drug for tauopathies. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388066v1?rss=1 Authors: Longhan, X., Wang, Z., Huang, G., Liu, B., Zhou, Z., Liang, B., Liu, Y. Abstract: Lower limb assistive robots have a wide range of applications in medical rehabilitation, hiking, and the military. The purpose of this work is to investigate the efficiency of wearable assistive devices under different weight-bearing walking conditions. We designed an experimental platform, with a lightweight ankle-assisted robot weighing 5.2 kg and carried mainly on the back. Eight subjects were tested in three experimental conditions: free walk with load (FWL), power-off with load (POFL), and power-on with load (PONF) for different levels of force at a walking speed of 3.6 km/h. We recorded the metabolic expenditure and kinematics of the subjects under three levels of weight-bearing (equal to 10%, 20%, and 30% of body mass). The critical forces from the fit of the assistive force and metabolic depletion curves were 130 N, 160 N and 215 N at three different load levels. The intrinsic weight of our device increases mechanical work at the ankle as the load weight rises, with 2.08 J, 2.43 J, 2.73 J for one leg during a gait cycle. The ratio of the mechanical work input by the robot to the mechanical work output by the weight of the device decreases (0.904, 0.717, and 0.513 with different load carriages), verifying that the walking assistance efficiency of such devices decreases as the weight rises. In terms of mechanical work in the ankle joint, our results confirm that the efficiency of the ankle-assisted walking robot decreases as weight bearing increases, which provides important guidance for the lightweight design of portable weight-bearing walking robots. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390179v1?rss=1 Authors: Pohorille, A., Wei, C. Abstract: To understand the transition from inanimate matter to life, we studied a process that directly couples simple metabolism to evolution via natural selection, demonstrated experimentally by Adamala and Szostak (Nat. Chem. 2013, 5, 495-501). In this process, dipeptides synthesized inside precursors of cells promote absorption of fatty acid micelles to vesicles inducing their preferential growth and division at the expense of other vesicles. The process is explained on the basis of coarse-grained molecular dynamics simulations, each extending for tens of microseconds, carried out to model fusion between a micelle and a membrane, both made of fatty acids in the absence and presence of hydrophobic dipeptides. In all systems with dipeptides, but not in their absence, fusion events were observed. They involve the formation of a stalk made by hydrophobic chains from the micelle and the membrane, similar to that postulated for vesicle-vesicle fusion. The emergence of a stalk is facilitated by transient clusters of dipeptides, side chains of which formed hydrophobic patches at the membrane surface. Committor probability calculations indicate that the size of a patch is a suitable reaction coordinate and allow for identifying the transition state for fusion. Free energy barrier to fusion is greatly reduced in the presence of dipeptides to only 4-5 kcal/mol, depending of the hydrophobicity of side chains. The mechanism of mediated fusion, which is expected to apply to other small peptides and hydrophobic molecules, provides a robust means by which a nascent metabolism can confer evolutionary advantage to precursors of cells. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390161v1?rss=1 Authors: Jonely, M., Singh, R. K., Bass, B., Noriega, R. Abstract: Drosophila melanogaster Dicer-2 is a large, multidomain protein that cleaves double-stranded RNA (dsRNA) into small interfering RNAs in a terminus-dependent manner as part of the RNA interference pathway. We characterize the local binding environment involved in this substrate-selective molecular recognition event by monitoring the time-resolved photophysics of a cyanine dye linked to the dsRNA terminus. We observe substantial changes in the molecular rigidity and local freedom of motion of the probe as a function of distinct conformations of the biomolecular complex between Dicer-2 and dsRNA as a function of dsRNA termini, the presence of regulatory proteins, and the addition of a biochemical energy source (ATP) or a non-hydrolysable equivalent (ATP-{gamma}S). With a clustering analysis based solely on these molecular-scale measures of the local binding environment at the dsRNA terminus, we identify sub-populations of similar conformations that define distinct modes of molecular recognition which are correlated with biochemical activity. These observations reveal the important role of substrate-selective molecular recognition properties for proteins with multiple domains that can bind RNA, regulatory proteins, and cofactors. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.388447v1?rss=1 Authors: Anselmi, M., Hub, J. S. Abstract: SHP2 is a critical regulator of signal transduction implicated in developmental disorders and cancer. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as a key "allosteric switch" for SHP2 activation. We critically review structural data and use extensive molecular simulations to test the "allosteric switch" model of activation. However, we find that the binding cleft in N-SH2 is constitutively flexible and open in solution, and that a closed cleft found in certain structures has been imposed by crystal contacts. The free energy cost for N-SH2 release is only marginally influenced by the binding cleft. We conclude that not the N-SH2 binding cleft but instead the opening of a central {beta}-sheet of N-SH2 is the key allosteric switch triggering SHP2 activation. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.384313v1?rss=1 Authors: Mazal, H. A., Iljina, M., Riven, I., Haran, G. Abstract: AAA+ ring-shaped machines, such as ClpB and Hsp104, mediate substrate translocation through their central channel by a set of pore loops. Recent structural studies suggested a universal hand-over-hand translocation mechanism, in which pore loops are moving rigidly in tandem with their corresponding subunits. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB and their response to substrate binding, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond timescale, and change their conformation upon interaction with substrate proteins. Pore-loop conformational dynamics are modulated by nucleotides and strongly correlate with disaggregation activity. The differential behavior of the pore loops along the axial channel points to a fast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.389775v1?rss=1 Authors: Uematsu, M., Shimizu, T., Shindou, H. Abstract: The physical properties of lipids, such as viscosity, are homeostatically maintained in cells, and are intimately involved in physiological roles. Measurement of the physical properties of plasma membranes has been achieved primarily through chemical or genetically encoded fluorescent probes, however, the effect of the probes themselves on these physical properties hampered accurate measurements. In addition, as most probes target plasma membranes, physical properties of lipids in intracellular organelles, including lipid droplets (LDs) are yet to be analyzed. Here, we present a novel Raman microscopy-based approach for quantifying the intracellular physical properties of lipids under deuterium-labeled fatty acid treatment conditions. Focusing on the fact that Raman spectra of carbon-deuterium vibration are altered depending on the surrounding lipid species, we quantitatively represented the physical properties of lipids as the gauche/trans conformational ratio of the introduced labeled fatty acids, which can be used as an indicator of viscosity. Intracellular Raman imaging revealed that the gauche/trans ratio of cytosolic regions was robustly preserved against stimuli attempting to alter the lipid composition. This was likely due to LDs functioning as a buffer against excess gauche/trans ratio. The novel approach, that is, using the target lipid itself as a probe, overcomes the issues presented by conventional probes, making it useful for the quantitative evaluation of biological functions and regulatory mechanisms associated with the physical state of intracellular lipid environments. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.389494v1?rss=1 Authors: Shan, Y., Zhang, X., Song, C., Liu, Y., Ke, P., Kuo, Y.-C., Wang, Y. Abstract: Plexins are semaphorin receptors that play essential roles in neuronal axon guidance and in many other important biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism, and is modulated by small signaling GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations we showed that RND1 reinforces plexin dimerization interface whereas RhoD destabilizes it due to their differential interaction with cell membrane. Upon binding plexin dimers at the Rho-GTPase binding (RBD) domains, RND1 and RhoD interact differently with the inner leaflet of cell membrane, and exert opposite effects on the dimerization interface via an allosteric network involving the RBD domain, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively-charged membrane interface. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.383745v1?rss=1 Authors: Reyes-Aldrete, E., Dill, E., Bussetta, C., Szymanski, M. R., Diemer, G. S., Maindola, P., White, M., Choi, K., Morais, M. Abstract: Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here we describe biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage ascc{varphi}28. Size-exclusion chromatography, analytical ultracentrifugation, small angle x-ray scattering, and negative stain TEM indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Angstroms and radius of gyration of ~54 Angstroms. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other dsDNA phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high resolution structural studies and rigorous biophysical/biochemical analysis. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.386524v1?rss=1 Authors: Hanson, B. S., Read, D. J. Abstract: Many biophysical systems and proteins undergo mesoscopic conformational changes in order to perform their biological function. However, these conformational changes often result from a cascade of atomistic interactions within a much larger overall object. For simulations of such systems, the computational cost of retaining high-resolution structural and dynamical information whilst at the same time observing large scale motions over long times is high. Furthermore, this cost is only going to increase as ever larger biological objects are observed experimentally at high resolution. With insight from the theory of Markov state models and transition state theory, we derive a generalised mechano-kinetic simulation framework which aims to compensate for these disparate time scales. The framework models continuous dynamical motion at coarse-grained length scales whilst simultaneously including fine-detail, chemical reactions or conformational changes implicitly using a kinetic model. The kinetic model is coupled to the dynamic model in a highly generalised manner, such that it can be applied to any defined continuous energy landscape, and indeed, any existing dynamic simulation framework. We present a series of analytical examples to validate the framework, and showcase its capabilities for studying more complex systems by designing a simulation of minimalist molecular motor. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387662v1?rss=1 Authors: Johnson, S. L., Narlikar, G. Abstract: ATP-dependent chromatin remodelers are essential enzymes that restructure eukaryotic genomes to enable all DNA-based processes. The diversity and complexity of these processes are matched by the complexity of the enzymes that carry them out, making remodelers a challenging class of molecular motors to study by conventional methods. Here we use a single molecule biophysical assay to overcome some of these challenges, enabling a detailed mechanistic dissection of a paradigmatic remodeler reaction, that of sliding a nucleosome towards the longer DNA linker. We focus on how two motors of a dimeric remodeler coordinate to accomplish such directional sliding. We find that ATP hydrolysis by both motors promotes coordination, suggesting a role for ATP in resolving the competition for directional commitment. Furthermore, we show an artificially constitutive dimer is no more or less coordinated, but is more processive, suggesting a cell could modulate a remodeler's oligomeric state to modulate local chromatin dynamics. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.18.365965v1?rss=1 Authors: Haloi, N., Wen, P.-C., Cheng, Q., Yang, M., Natarajan, G., Camara, A. K. S., Kwok, W.-M., Tajkhorshid, E. Abstract: Complex formation between hexokinase-II (HKII) and the mitochondrial channel VDAC1 plays a crucial role in regulating cell growth and survival; however, structural details of this complex remain elusive. We hypothesize that a conserved, hydrophobic helix (H-anchor) of HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we adopted a hybrid approach: 1) the membrane binding of HKII was first described with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion, then 2) the resulting membrane-bound HKII was used to form complex with VDAC1 in millisecond-scale Brownian dynamics (BD) simulations. We show that H-anchor inserts its first 10 residues into the membrane, substantiating previous experimental findings. The insertion depth of the H-anchor was used to derive positional restraints in subsequent BD simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models were further refined with MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1 permeation pathway by HKII, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. Additionally, we showed how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and have implications in mitochondria-mediated cell death. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384958v1?rss=1 Authors: Blosser, M. C., So, J., Madani, M. S., Malmstadt, N. Abstract: Determining the permeability of lipid membranes to gases is important for understanding the biological mechanisms of gas transport. Experiments on model membranes have been used to determine the permeability of lipid bilayers in the absence of proteins. Previous measurements have used a number of different methods and obtained widely varying results. We have developed a microfluidic based microscopy assay that measures the rate of CO2 permeation in Giant Unilamellar Vesicles (GUVs), and we report permeability data for the POPC-cholesterol system. We find that cholesterol has a strong effect on permeability; bilayers containing high levels of cholesterol are an order of magnitude less permeable than bilayers without cholesterol, 9.9 {+/-} 1.0 x 10-4 cm/s vs. 9.6 {+/-} 1.4 x 10-3 cm/s. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384602v1?rss=1 Authors: Vanhille Campos, C., Saric, A. Abstract: We study the effects of osmotic shocks on lipid vesicles via coarse-grained molecular dynamics simulations by explicitly considering the solute in the system. We find that depending on their nature (hypo- or hypertonic) such shocks can lead to bursting events or engulfing of external material into inner compartments, among other morphology transformations. We characterize the dynamics of these processes and observe a separation of time scales between the osmotic shock absorption and the shape relaxation. Our work consequently provides an insight into the dynamics of compartmentalization in vesicular systems as a result of osmotic shocks, which can be of interest in the context of early proto-cell development and proto-cell compartmentalisation. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384727v1?rss=1 Authors: Meisl, G., Zuo, Y., Allinson, K., Rittman, T., DeVos, S., Sanchez, J. S., Xu, C. K., Duff, K. E., Johnson, K. A., Rowe, J., Hyman, B., Knowles, T. P., Klenerman, D. Abstract: Both the replication of protein aggregates and their spreading throughout the brain are implicated in the progression of Alzheimer's disease (AD). However, the rates of these processes are unknown and the identity of the rate-determining process in humans has therefore remained elusive. By bringing together chemical kinetics with measurements of tau seeds and aggregates across brain regions, we are able to quantify their replication rate in human brains. Remarkably, we obtain comparable rates in several different datasets, with 5 different methods of tau quantification, from seed amplification assays in vitro to tau PET studies in living patients. Our results suggest that the overall rate of accumulation of tau in neocortical regions is limited not by spreading between brain regions but by local replication, which doubles the number of seeds every ~5 years. Thus, we propose that limiting local replication constitutes the most promising strategy to control tau accumulation during AD. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.384248v1?rss=1 Authors: Ball, S. R., Adamson, J. S., Sullivan, M. A., Zimmermann, M. R., Lo, V., Sanz-Hernandez, M., Jiang, F., Kwan, A. H., Werry, E. L., Knowles, T. P., Kassiou, M., Meisl, G., Todd, M. H., Rutledge, P. R., Sunde, M. Abstract: The amyloid-{beta} peptide, the main protein component of amyloid plaques in Alzheimer's disease, plays a key role in the neurotoxicity associated with the condition through the formation of small toxic oligomer species which mediate the disruption of calcium and glutamate homeostasis. The lack of therapeutic benefit associated with removal of mature amyloid-{beta} fibrils has focused attention on the toxic oligomeric species formed during the process of fibril assembly. Here, we present the design and synthesis of a family of perphenazine-macrocyle conjugates. We find that two-armed perphenazine-cyclam conjugates divert the monomeric form of the amyloid-{beta} peptide away from the amyloidogenic pathway into amorphous aggregates that are not toxic to differentiated SH-SY5Y cells in vitro. This strategy prevents the formation of damaging amyloid oligomers. Kinetic analysis of the effects of these compounds on the assembly pathway, together with NMR spectroscopy, identifies rapid monomer sequestration as the underlying neuroprotective mechanism. The ability to specifically target the monomeric form of amyloid-{beta} allows for further understanding of the impact of the multiple species formed between peptide biogenesis and plaque deposition. The modular, three-dimensional structure of these compounds provides a starting point for the design of more potent modulators of this amyloid-forming peptide, and can be adapted to probe the protein self-assembly pathways associated with other proteinopathies. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.15.383794v1?rss=1 Authors: Kapoor, k., Pant, S., Tajkhorshid, E. Abstract: P-glycoprotein (Pgp) is a major efflux pump in humans, overexpressed in a variety of cancers and associated with the development of multi-drug resistance. Allosteric modulation induced by binding of various ligands (e.g., transport substrates, inhibitors, and ATP) has been bio-chemically shown to directly influence the function of Pgp. However, the molecular details of such effects are not well established. In particular, the role and involvement of the sur-rounding lipid environment on ligand-induced modulation of the conformational dynamics of the transporter have not been investigated at any level. Here, we employ all-atom molecular dynamics (MD) simulations to study the conformational landscape of Pgp in the presence of a high-affinity, third-generation inhibitor, tariquidar, in comparison to the nucleotide-free (APO) and the ATP-bound states, in order to shed light on and to characterize how the inhibitor blocks the function of the transporter. Simulations in a multi-component lipid bi-layer show a dynamic equilibrium between open and closed inward-facing (IF) conformations in the APO-state, with binding of ATP shifting the equilibrium towards conformations feasible for ATP hydrolysis and subsequent completion of the transport cycle. In the presence of the inhibitor bound to the drug-binding pocket in the transmembrane domain (TMD), the transporter samples more open IF conformations, and the nucleotide binding domains (NBDs) are observed to become highly dynamic. Interestingly, and reproduced in multiple independent simulations, the inhibitor is observed to recruit lipid molecules into the Pgp lumen through the two proposed drug-entry portals, where the lipid head groups from the lower leaflet translocate inside the TMD, while the lipids tails remain extended into the bulk lipid environment. These 'wedge-lipid' molecules likely enhance the inhibitor-induced conformational changes in the TMD leading to the differential modulation of coupling pathways observed with the NBDs downstream. We suggest a novel inhibitory mechanism for tariquidar, and for related third-generation Pgp inhibitors, where lipids are seen to enhance the inhibitory role in the catalytic cycle of membrane transporters. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.382242v1?rss=1 Authors: Lau, D., Walsh, J. C., Dickson, C. F., Tuckwell, A., Stear, J. H., Hunter, D. J. B., Bhumkar, A., Shah, V., Turville, S. G., Sierecki, E., Gambin, Y., Jacques, D. A., Böcking, T. Abstract: The HIV capsid is a multifunctional protein capsule for delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labelled capsid-binding analytes ( 'prey' molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using fluorescent capsid as bait further allows quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for discovery and characterization of molecules used by HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.15.383729v1?rss=1 Authors: Rosencrans, W. M., Aguilella, V. M., Rostovtseva, T. K., Bezrukov, S. M. Abstract: When the Parkinson's disease (PD) related neuronal protein, alpha-synuclein (Syn), is added to the reconstituted mitochondrial voltage-dependent anion channel (VDAC), it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule electrophysiology of reconstituted VDAC we now demonstrate that, at CaCl2 concentrations below 150 mM, Syn reverses the channel's selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by Syn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. These findings reveal a new regulatory role of Syn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.382176v1?rss=1 Authors: Mitteaux, J., Lejault, P., Pirrotta, M., Wojciechowski, F., Joubert, A., Desbois, N., Gros, C. P., Hudson, R. H. E., Boule, J.-B., Granzhan, A., Monchaud, D. Abstract: The quest for small molecules that avidly bind to G-quadruplex-DNA (G4-DNA, or G4), so called G4-ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to i- screen or design G4-ligands, ii- evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and iii- use them as unique chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts at developing small molecules aimed at destabilizing G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application to neurobiology as many genetic and age-related diseases are caused by an over-representation of G4s, itself caused by a deficiency of G4-resolving enzymes, the G4-helicases. Herein, we report on our double effort to i- develop a reliable in vitro assay to identify molecules able to destabilize G4s, the G4-unfold assay, and ii- fully characterize the first prototype of G4-disrupting small molecule, a phenylpyrrolcytosine (PhpC)-based G-clamp analog. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.14.381376v1?rss=1 Authors: Zhang, Y., Wright, M., Saar, K. L., Challa, P., Morgunov, A. S., Peter, Q. A. E., Devenish, S., Dobson, C. M., Knowles, T. P. J. Abstract: The ability to determine the identity of specific proteins is a critical challenge in many areas of cellular and molecular biology, and in medical diagnostics. Here, we present a microfluidic protein characterisation strategy that within a few minutes generates a three-dimensional fingerprint of a protein sample indicative of its amino acid composition and size and, thereby, creates a unique signature for the protein. By acquiring such multidimensional fingerprints for a set of ten proteins and using machine learning approaches to classify the fingerprints, we demonstrate that this strategy allows proteins to be classified at a high accuracy, even though classification using a single dimension is not possible. Moreover, we show that the acquired fingerprints correlate with the amino acid content of the samples, which makes it is possible to identify proteins directly from their sequence without requiring any prior knowledge about the fingerprints. These findings suggest that such a multidimensional profiling strategy can lead to the development of novel method for protein identification in a microfluidic format. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381897v1?rss=1 Authors: Zeng, X., Liu, C., Fossat, M. J., Ren, P., Chilkoti, A., Pappu, R. V. Abstract: Many naturally occurring elastomers are intrinsically disordered proteins (IDPs) built up of repeating units and they can demonstrate two types of thermoresponsive phase behavior. Systems characterized by lower critical solution temperatures (LCST) undergo phase separation above the LCST whereas systems characterized by upper critical solution temperatures (UCST) undergo phase separation below the UCST. There is congruence between thermoresponsive coil-globule transitions and phase behavior. Specifically, the theta temperatures above or below which the IDPs transition from coils to globules serve as useful proxies for the LCST / UCST values. This implies that one can design sequences with desired values for the theta temperature with either increasing (UCST) or decreasing radii of gyration (LCST) above the theta temperature. Here, we show that the Monte Carlo simulations performed in the so-called intrinsic solvation (IS) limit version of the temperature-dependent ABSINTH implicit solvation model, yields a robust heuristic for discriminating between sequences with known LCST versus UCST phase behavior. Accordingly, we use this heuristic in a supervised approach, integrate it with a genetic algorithm, combine this with IS limit simulations, and show how novel sequences can be designed that have LCST phase behavior. These calculations are aided by direct estimates of temperature dependent free energies of solvation for model compounds that are derived using the polarizable AMOEBA forcefield. To demonstrate the validity of our designs, we calculate coil-globule transition profiles using the full ABSINTH model and combine these with the Gaussian Cluster Theory to show that the designed IDPs do show LCST phase behavior. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.382218v1?rss=1 Authors: Hosseinizadeh, A., Breckwoldt, N., Fung, R., Sepehr, R., Schmidt, M., Schwander, P., Santra, R., Ourmazd, A. Abstract: The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate energy surfaces. Posited almost a century ago [1], conical intersections remain the subject of intense scientific investigation [2-4]. In biology, they play a pivotal role in vision, photosynthesis, and DNA stability [5,6]. In ultrafast radiationless de-excitation [1,7], they are vital to ameliorating photon-induced damage. In chemistry, they tightly couple the normally separable nuclear and electronic degrees of freedom, precluding the Born-Oppenheimer approximation [8]. In physics, they manifest a Berry phase, giving rise to destructive interference between clockwise and anti-clockwise trajectories around the conical intersection [9]. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around 10 atoms, on timescales of about 100 fs or longer [10]. Spectroscopy can achieve better time resolution, but provides only indirect structural information. Here, we present single-femtosecond, atomic-resolution movies of a 2,000-atom protein passing through a conical intersection. These movies, extracted from experimental data by geometric machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process, and elucidate the topography of the electronic potential energy surfaces involved. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381152v1?rss=1 Authors: Gebhardt, C., Lehmann, M., Reif, M., Zacharias, M., Cordes, T. Abstract: The use of fluorescence techniques has had an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the availability of numerous commercial dyes, but often information about the chemical structures of dyes (and their linkers) are a well-kept secret. This can lead to problems for applications where a knowledge of the dye structure is necessary to predict (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of spectroscopy, mass spectrometry and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we present two possible structures for Alexa Fluor 555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647. A comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes, supports the suggestion that the Alexa Fluor and AF dyes have a high degree of structural similarity. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation demonstrating that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes for smFRET studies, and suggesting their usefulness for other fluorescence applications. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.379982v1?rss=1 Authors: Thiel, G., Tandl, D. H., Sponagel, T., Fuck, S., Smit, T., Hehlgans, S., Jakob, B., Fournier, C., Roedel, F., Roth, B., Moroni, A. Abstract: Radiation therapy is efficiently employed for eliminating cancer cells and reducing tumor growth. To further improving its therapeutic application it is mandatory to unravel the molecular effects of ionizing irradiation and to understand whether they support or counteract tumor therapy. Here we examine the impact of X-ray irradiation on immune activation of human T cells with single doses typically employed in tumor therapy. We discover that exposing cells to radiation triggers in a population of leukemic Jurkat T cells and in peripheral blood mononuclear cells (PBMCs) a canonical Ca2+ signaling cascade, which elicits immune activation of these cells. An early step in the signaling cascade is the initiation of sustained oscillations of the cytosolic Ca2+ concentration, an event mediated by store operated Ca2+ entry (SOCE) via an X-ray induced clustering of the Calcium Release-Activated Calcium Modulator 1 with the stromal interaction molecule 1 (Oari1/STIM1). A functional consequence of the Ca2+ signaling cascade is the translocation of the transcription factor nuclear factor of activated T cells (NFAT) from the cytosol into the nucleus where it elicits the expression of genes required for immune activation. These data imply that a direct activation of blood immune cells by ionizing irradiation has an impact on toxicity and therapeutic effects of radiation therapy. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381632v1?rss=1 Authors: Barrera, E. E., Zonta, F., Pantano, S. Abstract: Poly glutamine and glutamine-rich peptides play a central role in a plethora of pathological aggregation events. However, biophysical characterization of soluble oligomers --the most toxic species involved in these processes-- remains elusive due to their structural heterogeneity and dynamical nature. Here, we exploit the high spatio-temporal resolution of simulations as a computational microscope to characterize the aggregation propensity and morphology of a series of polyglutamine and glutamine-rich peptides. Comparative analysis of ab-initio aggregation pinpointed a double role for glutamines. In the first phase, glutamines mediate seeding by pairing monomeric peptides, which serve as primers for higher-order nucleation. According to the glutamine content, these low molecular-weight oligomers may then proceed to create larger aggregates. Once within the aggregates, buried glutamines continue to play a role in their maturation by optimizing solvent-protected hydrogen bonds networks. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.382036v1?rss=1 Authors: Monsen, R. C., Chakravarthy, S., Dean, W. L., Chaires, J. B., Trent, J. O. Abstract: Human telomeres contain the repeat DNA sequence 5'(TTAGGG), with duplex regions that are several kilobases long terminating in a 3' single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate modes proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5'(TTAGGG)n, with n=8, 12, and 16, fold into multimeric structures containing the maximal number (2, 3, and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of about 34 angstroms. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.379479v1?rss=1 Authors: Kim, S. H., Kim, H., Jeong, H., Yoon, T.-Y. Abstract: DNA barcoding provides a way to label a huge number of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is an easy-to-access method to detect individual DNA barcodes, which can be scaled up to a massively high-throughput format. Large overlaps between emission spectra of fluorescence dyes, however, severely limit the numbers of DNA barcodes-and thus its signal space-that can be detected in a simultaneous manner. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak hybridization events for DNA barcodes, we markedly enhanced accuracy in our determination of the efficiency by which single-molecule FRET occurred, reaching an error less than 0.01 in the FRET efficiency domain. This allowed us to unambiguously differentiate six DNA barcodes exhibiting different FRET values without involving probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of the DNA barcode techniques. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.379065v1?rss=1 Authors: Araujo, T. S., Scapin, S. M., Andrade, W., Fasciotti, M., de Magalhaes, M. T., Almeida, M. S., Lima, L. M. T. d. R. Abstract: The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.379230v1?rss=1 Authors: Nakai, T., Ando, T., Goto, T. Abstract: Many kinds of peritrichous bacteria that repeat runs and tumbles by using multiple flagella exhibit chemotaxis by sensing a difference in the concentration of the attractant or repellent between two adjacent time points. If a cell senses that the concentration of an attractant has increased, their flagellar motors decrease the switching frequency from counterclockwise to clockwise direction of rotation, which causes a longer run in swimming up the concentration gradient than swimming down. We investigated the turn angle in tumbles of peritrichous bacteria swimming across the concentration gradient of a chemoattractant because the change in the switching frequency in the rotational direction may affect the way tumbles. We tracked several hundreds of runs and tumbles of single Salmonella typhimurium cells in the concentration gradient of L-serine, and found that the turn angle depends on the concentration gradient that the cell senses just before the tumble. The turn angle is biased toward a smaller value when the cells swim up the concentration gradient, whereas the distribution of the angle is almost uniform (random direction) when the cells swim down the gradient. The effect of the observed bias in the turn angle on the degree of chemotaxis was investigated by random walk simulation. In the concentration field where attractants diffuse concentrically from the point source, we found that this angular distribution clearly affects the reduction of the mean square displacement of the cell that has started at the attractant source, that is, the bias in the turn angle distribution contributes to chemotaxis in peritrichous bacteria. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378828v1?rss=1 Authors: Niemi, A. J., Peng, X. Abstract: The spike protein is a most promising target for the development of vaccines and therapeutic drugs against the SARS-CoV-2 infection. But the apparently high rate of mutations makes the development of antiviral inhibitors a challenge. Here a methodology is presented to try and predict mutation hot-spot sites, where a small local change in spike protein's structure can lead to a large scale conformational effect, and change the protein's biological function. The methodology starts with a systematic physics based investigation of the spike protein's C backbone in terms of its local topology. This topological investigation is then combined with a statistical examination of the pertinent backbone fragments; the statistical analysis builds on a comparison with high resolution Protein Data Bank (PDB) structures. Putative mutation hot-spot sites are identified as proximal sites to bifurcation points that can change the local topology of the C backbone in an essential manner. The likely outcome of a mutation, if it indeed occurs, is predicted by a comparison with residues in best-matching PDB fragments together with general stereochemical considerations. The detailed methodology is developed using the already observed D614G mutation as an example. This is a mutation that could have been correctly predicted by the present approach. Several additional examples of potential hot-spot residues are identified and analyzed in detail, some of them are found to be even better candidates for a mutation hot-spot than D614G. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.378422v1?rss=1 Authors: Guenther, S., Reinke, P. Y. A., Fernandez-Garcia, Y., Lieske, J., Lane, T. J., Ginn, H., Koua, F., Ehrt, C., Ewert, W., Oberthuer, D., Yefanov, O., Meier, S., Lorenzen, K., Krichel, B., Kopicki, J., Gelisio, L., Brehm, W., Dunkel, I., Seychell, B., Gieseler, H., Norton-Baker, B., Escudero-Perez, B., Domaracky, M., Saouane, S., Tolstikova, A., White, T., Haenle, A., Groessler, M., Fleckenstein, H., Trost, F., Galchenkova, M., Gevorkov, Y., Li, C., Awel, S., Peck, A., Barthelmess, M., Schluenzen, F., Lourdu, X. P., Werner, N., Andaleeb, H., Ullah, N., Falke, S., Srinivasan, V., Franca, B., Schwi Abstract: The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.375105v1?rss=1 Authors: Skrbic, T., Maritan, A., Giacometti, A., Rose, G. D., Banavar, J. R. Abstract: The native state structures of globular proteins are stable and well-packed indicating that self-interactions are favored over protein-solvent interactions under folding conditions. We use this as a guiding principle to derive the geometry of the building blocks of protein structures, alpha-helices and strands assembled into beta-sheets, with no adjustable parameters, no amino acid sequence information, and no chemistry. There is an almost perfect fit between the dictates of mathematics and physics and the rules of quantum chemistry. Our theory establishes an energy landscape that channels protein evolution by providing sequence-independent platforms for elaborating sequence-dependent functional diversity. Our work highlights the vital role of discreteness in life and has implications for the creation of artificial life and on the nature of life elsewhere in the cosmos. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378638v1?rss=1 Authors: Ilangumaran Ponmalar, I., Ayappa, K. G., Basu, J. K. Abstract: Developing alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors require an understanding of the target cellular response to combat rising antimicrobial resistance. Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane disruption and related lipid mediated active repair processes during attack by PFTs remain largely unexplored. We report counter intuitive and non-monotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane bound oligomers of a prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO). The observed protein concentration dependent dynamical cross-over is correlated with transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events due to a preponderance of ring-like pore states. At higher protein concentrations where membrane inserted arc-like pores dominate, lipid ejection is less eficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodelling and mechanical property variations, providing unique insights into developing strategies to combat virulent bacterial pathogens responsible for several infectious diseases. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378026v1?rss=1 Authors: Suvlu, D., Thirumalai, D., Rasaiah, J. C. Abstract: Water-mediated interactions (WMIs) play diverse roles in molecular biology. They are particularly relevant in geometrically confined spaces such as the interior of the chaperonin, at the interface between ligands and their binding partners, and in the ribosome tunnel. Inspired in part by the geometry of the ribosome tunnel, we consider confinement effects on the stability of peptides. We describe results from replica exchange molecular dynamics simulations of a system containing a 23-alanine or 23-serine polypeptide confined to non-polar and polar nanotubes in the gas phase and when open to a water reservoir. We quantify the effect of water in determining the preferred conformational states of these polypeptides by calculating the difference in the solvation free energy for the helix and coil states in the open nanotube in the two phases. Our simulations reveal several possibilities. We find that nanoscopic confinement preferentially stabilizes the helical state of polypeptides with hydrophobic side chains, which is explained by the entropic stabilization mechanism proposed on the basis of polymer physics. Polypeptide chains with hydrophilic side chains can adopt helical structures within nanotubes, but helix formation is sensitive to the nature of the nanotube due to WMIs. We elaborate on the potential implications of our findings to the stability of peptides in the ribosome tunnel. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.377200v1?rss=1 Authors: Carlson, M. L., Seyler, S. L., Presse, S. Abstract: Bdellovibrio bacteriovorus is a predatory bacterium that preys upon gram-negative bacteria. As such, B. bacteriovorus has the potential to control antibiotic-resistant pathogens and biofilm populations. To survive and reproduce, B. bacteriovorus must locate and infect a host cell. However, in the temporary absence of prey, it is largely unknown how B. bacteriovorus modulate their motility patterns in response to physical or chemical environmental cues to optimize their energy expenditure. To investigate B. bacteriovorus' predation strategy, we track and quantify their motion by measuring speed distributions and velocity autocorrelations as a function of starvation time. An initial unimodal speed distribution, relaxing to that expected for pure diffusion at long times, may be expected. Instead, we observe a complex, non-Brownian, search strategy as evidenced by distinctly bimodal speed distributions. That is, for an increasing amount of time over which B. bacteriovorus is starved, we observe a progressive re-weighting from a fast mode to a slow mode in the speed distribution obtained over consecutive frames. By contrast to its predator, B. bacteriovorus' prey, Escherichia coli exhibits almost immediate decrease to a speed expected from passive diffusion following resuspension from rich to poor media. Distributions of trajectory-averaged speeds for B. bacteriovorus are largely unimodal, indicating nontrivial switching between fast and slow swimming modes within individual observed trajectories rather than there being distinct fast and slow populations. We also find that B. bacteriovorus' slow speed mode is not merely caused by the diffusion of inviable bacteria as subsequent spiking experiments show that bacteria can be resuscitated and bimodality restored. Indeed, starved B. bacteriovorus may modulate the frequency and duration of active swimming as a means of balancing energy consumption and procurement. Our results are evidence of a nontrivial predation strategy, which contrasts with the comparatively simple search pattern of its prey, in response to environmental cues. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.11.378646v1?rss=1 Authors: Lee, W. H., Liu, W., Fan, J.-S., Yang, D. Abstract: The viral protease domain (NS3pro) of dengue virus is essential for virus replication and its cofactor NS2B is indispensable for the proteolytic function. Although several NS3pro-NS2B complex structures have been obtained, the dynamic property of the complex remains poorly understood. Using NMR relaxation techniques, here we found that NS3pro-NS2B exists in both closed and open conformations which are in dynamic equilibrium on a sub-millisecond timescale in aqueous solution. Our structural information indicates that the C-terminal region of NS2B is disordered in the open conformation but folded in the closed conformation. Using mutagenesis, we showed that the closed-open conformational equilibrium can be shifted by changing NS2B stability. Moreover, we revealed that the proteolytic activity of NS3pro-NS2B correlates well with the population of the closed conformation. Our results suggest that the closed-open conformational equilibrium can be used by both nature and man to control the replication of dengue virus. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.376285v1?rss=1 Authors: Brotzakis, Z. F., Lindstedt, P., Taylor, R., Bernardes, G., Vendruscolo, M. Abstract: Tau is a microtubule-associated protein that regulates the stability of microtubules. The affinity of tau for microtubules is modulated by post-translational modifications, and the dysregulation of these events has been associated with the aberrant aggregation of tau in Alzheimer's disease and related tauopathies. Here, we use the metainference cryo- electron microscopy approach to determine an ensemble of structures representing the structure and dynamics of a tau-microtubule complex comprising an extended microtubule-binding region of tau (residues 202-395). We thus identify the ground state of the complex and a series of excited states of lower populations. An analysis of the interactions in these states of structures reveals positions in the tau sequence that are important to determine the overall stability of the tau-microtubule complex. This analysis leads to the identification of positions where phosphorylation and acetylation events have destabilising effects, which we validate by using site-specific post-translationally modified tau variants obtained by chemical mutagenesis. Taken together, these results illustrate how the simultaneous determination of ground and excited states of macromolecular complexes reveals functional and regulatory mechanisms. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.10.375493v1?rss=1 Authors: Vander Roest, A. S., Liu, C., Morck, M. M., Kooiker, K. B., Jung, G., Song, D., Dawood, A., Jhingran, A., Pardon, G., Ranjbarvaziri, S., Fajardo, G., Zhao, M., Campbell, K. S., Pruitt, B., Spudich, J. A., Ruppel, K. M., Bernstein, D. Abstract: Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, many in {beta}-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreases in vitro motility and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load-sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super-relaxed state in larger, two-headed myosin constructs, freeing more heads to generate force. Micropatterned hiPSC-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling, as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrates measured molecular changes to demonstrate that predicted force traces match the forces measured in cells. These results confirm a key role for regulation of the super-relaxed state in driving hypercontractility in HCM and demonstrate the value of a multiscale approach in revealing key mechanisms of disease. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.375691v1?rss=1 Authors: Stadlbauer, P., Islam, B., Otyepka, M., Chen, J., monchaud, d., Zhou, J., Mergny, J.-L., Sponer, J. Abstract: Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicit-solvent molecular dynamics simulations to scrutinize the G4/hemin interaction. We found that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. Importantly, the simulations do not support several mechanistic possibilities (i.e., the wobbling guanine and the iron-bound water molecule) but rather suggest tentative mechanisms in which the external G-quartet itself is responsible for the unique H2O2-promoted biocatalytic properties of the G4/hemin complexes. Our simulations show that once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD simulation technique are also briefly discussed. Copy rights belong to original authors. Visit the link for more info
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