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bioRxiv2018

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. How the highly intertwined structure of 60S large ribosomal subunits is established is unknown. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 [A] resolution, revealing a half assembled subunit. Domains I, II and VI of 25S/5.8S rRNA tightly pack into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to the establishment of the global architecture of 60S subunits.

biophysics↗

bioRxiv2018

The extent of B-form structure in a 6S RNA variant that mimics DNA to bind to the Escherichia coli RNA Polymerase

In a recent article by Darst and coworkers, it was found that a non-coding 6S RNA variant regulates a bacterial RNA polymerase by mimicking B-Form DNA, and a few different nucleic acid duplex parameters were analyzed to understand the extent of B-form RNA structure. In this manuscript, a different structural analysis based on conformational distance from canonical A-form and B-form single-strand structures is presented. This analysis addresses the occurrence and extent of both local and global B-form structure in the published 6S RNA variant model.

biophysics↗

bioRxiv2018

Universal temporal rate of DNA replication origin firing: A balance between origin activation and passivation

The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris. We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics.

biophysics↗

bioRxiv2018

Heterochromatin drives organization of conventional and inverted nuclei

The mammalian cell nucleus displays a remarkable spatial segregation of active euchromatic from inactive heterochromatic genomic regions. In conventional nuclei, euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. In contrast, rod photoreceptors in nocturnal mammals have inverted nuclei, with a dense heterochromatic core and a thin euchromatic outer shell. This inverted architecture likely converts rod nuclei into microlenses to facilitate nocturnal vision, and may relate to the absence of particular proteins that tether heterochromatin to the lamina. However, both the mechanism of inversion and the role of interactions between different types of chromatin and the lamina in nuclear organization remain unknown. To elucidate this mechanism we performed Hi-C and microscopy on cells with inverted nuclei and their conventional counterparts. Strikingly, despite the inversion evident in microscopy, both types of nuclei display similar Hi-C maps. To resolve this paradox we developed a polymer model of chromosomes and found a universal mechanism that reconciles Hi-C and microscopy for both inverted and conventional nuclei. Based solely on attraction between heterochromatic regions, this mechanism is sufficient to drive phase separation of euchromatin and heterochromatin and faithfully reproduces the 3D organization of inverted nuclei. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. To further test our models, we eliminated lamina interactions in models of conventional nuclei and found that this triggers a spontaneous process of inversion that qualitatively reproduces the pathway of morphological changes during nuclear inversion in vivo. Together, our experiments and modeling suggest that interactions among heterochromatic regions are central to phase separation of the active and inactive genome in inverted and conventional nuclei, while interactions with the lamina are essential for building the conventional architecture from these segregated phases. Ultimately our data suggest that an inverted organization constitutes the default state of nuclear architecture.

biophysics↗

bioRxiv2018

Fidaxomicin jams M. tuberculosis RNA polymerase motions needed for initiation via RbpA contacts

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To enable structure-guided optimization of Fdx to treat tuberculosis, we report the 3.4 [A] cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin and explains its strong effect on M. tuberculosis. We present additional structures that define conformational states of M. tuberculosis RNAP between the free apo-holenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on Fdx.

biophysics↗

bioRxiv2018

Visualization of PRC2-Dinucleosome Interactions Leading to Epigenetic Repression

Epigenetic regulation is mediated by protein complexes that couple recognition of chromatin marks to activity or recruitment of chromatin-modifying enzymes. Polycomb repressive complex 2 (PRC2), a gene silencer that methylates lysine 27 of histone H3, is stimulated upon recognition of its own catalytic product, and has been shown to be more active on dinucleosomes than H3 tails or single nucleosomes. These properties likely facilitate local H3K27me2/3 spreading causing heterochromatin formation and gene repression. Here, cryo-EM reconstructions of human PRC2 bound to dinucleosomes show how a single PRC2, interacting with nucleosomal DNA, precisely positions the H3 tails to recognize a H3K27me3 mark in one nucleosome and is stimulated to modify a neighboring nucleosome. The geometry of the PRC2-DNA interactions allow PRC2 to tolerate different dinucleosome geometries due to varying lengths of the linker DNA. Our structures are the first to illustrate how an epigenetic regulator engages with a complex chromatin substrate.

biophysics↗

bioRxiv2018

Minimizing activation of overlying axons with epiretinal stimulation: The role of fiber orientation and electrode configuration

AO_SCPCAPBSTRACTC_SCPCAPObjective. Currently, a challenge in electrical stimulation of the retina is to excite only the cells lying directly under the electrode in the ganglion cell layer, while avoiding excitation of the axons that pass over the surface of the retina in the nerve fiber layer. Since these passing fibers may originate from distant regions of the ganglion cell layer. Stimulation of both target retinal ganglion cells and overlying axons results in irregular visual percepts, significantly limiting perceptual efficacy. This research explores how differences in fiber orientation between the nerve fiber layer and ganglion cell layer leads to differences in the activation of the axon initial segment and axons of passage. Approach. Axons of passage of retinal ganglion cells in the nerve fiber layer are characterized by a narrow distribution of fiber orientations, causing highly anisotropic spread of applied current. In contrast, proximal axons in the ganglion cell layer have a wider distribution of orientations. A four-layer computational model of epiretinal extracellular stimulation that captures the effect of neurite orientation in anisotropic tissue has been developed using a modified version of the standard volume conductor model, known as the cellular composite model. Simulations are conducted to investigate the interaction of neural tissue orientation, stimulating electrode configuration, and stimulation pulse duration and amplitude. Main results. The dependence of fiber activation on the anisotropic nature of the nerve fiber layer is first established. Via a comprehensive search of key parameters, our model shows that the simultaneous stimulation with multiple electrodes aligned with the nerve fiber layer can be used to achieve selective activation of axon initial segments rather than passing fibers. This result can be achieved with only a slight increase in total stimulus current and modest increases in the spread of activation in the ganglion cell layer, and is shown to extend to the general case of arbitrary electrode array positioning and arbitrary target neural volume. Significance. These results elucidate a strategy for more targeted stimulation of retinal ganglion cells with experimentally-relevant multi-electrode geometries and readily achievable stimulation requirements.

biophysics↗

bioRxiv2018

A mixed quantum chemistry/machine learning approach for the fast and accurate prediction of biochemical redox potentials and its large-scale application to 315,000 redox reactions

A quantitative understanding of the thermodynamics of biochemical reactions is essential for accurately modeling metabolism. The group contribution method (GCM) is one of the most widely used approaches to estimating standard Gibbs energies and redox potentials of reactions for which no experimental measurements exist. Previous work has shown that quantum chemical predictions of biochemical thermodynamics are a promising approach to overcome the limitations of GCM. However, the quantum chemistry approach is significantly more expensive. Here we use a combination of quantum chemistry and machine learning to obtain a fast and accurate method for predicting the thermodynamics of biochemical redox reactions. We focus on predicting the redox potentials of carbonyl functional group reductions to alcohols and amines, two of the most ubiquitous carbon redox transformations in biology. Our method relies on semi-empirical quantum chemistry calculations calibrated with Gaussian Process (GP) regression against available experimental data. Our approach results in higher predictive power than the GCM at a low computational cost. We design and implement a network expansion algorithm that iteratively reduces and oxidizes a set of natural seed metabolites, and demonstrate the high-throughput applicability of our method by predicting the standard potentials of more than 315,000 redox reactions involving approximately 70,000 compounds. Additionally, we developed a novel fingerprint-based framework for detecting molecular environment motifs that are enriched or depleted across different regions of the redox potential landscape. We provide open access to all source code and data generated.

biophysics↗

bioRxiv2018

Green function of correlated genes and themechanical evolution of protein

There has been growing evidence that cooperative interactions and configurational rearrangements underpin protein functions. But in spite of vast genetic and structural data, the information-dense, heterogeneous nature of protein has held back the progress in understanding the underlying principles. Here we outline a general theory of protein that quantitatively links sequence, dynamics and function: The protein is a strongly-coupled amino acid network whose interactions and large-scale motions are captured by the mechanical propagator, also known as the Green function. The propagator relates the gene to the connectivity of the amino acid network and the transmission of forces through the protein. How well the force pattern conforms to the collective modes of the functional protein is measured by the fitness. Mutations introduce localized perturbations to the propagator which scatter the force field. The emergence of function is manifested by a topological transition when a band of such perturbations divides the protein into subdomains. Epistasis quantifies how much the combined effect of multiple mutations departs from additivity. We find that epistasis is the nonlinearity of the Green function, which corresponds to a sum over multiple scattering paths passing through the localized perturbations. We apply this mechanical framework to the simulations of protein evolution, and observe long-range epistasis which facilitates collective functional modes. Our model lays the foundation for understanding the protein as an evolved state of matter and may be a prototype for other strongly-correlated living systems.

biophysics↗

bioRxiv2018

Chromatin configuration affects the dynamics and distribution of a transiently interacting protein

The interaction of proteins with chromatin regulates many cellular functions. Most DNA-binding proteins interact both non-specifically and transiently [1] with many chromatin sites as well as specifically and more stably with cognate binding sites. These interactions and chromatin structure are important in governing protein dynamics [2, 3]. However, the effect of these transient interactions on protein motion and distribution has yet been shown from a first principle.\n\nSome aspects of protein interactions with DNA have been studied in the context of the search of a gene promoter site by a transcription factor (TF) [4]. It was first noted [5] that the search for a promoter site by a TF would be faster if it involves 3d excursions, as well as sliding of the protein along DNA [6], as was shown in prokaryotes [7]. These different types of motion were observed ex ...

biophysics↗

bioRxiv2018

DETERMINATION OF THE THERMODYNAMICS OF β-LACTOGLOBULIN AGGREGATION USING ULTRA VIOLET LIGHT SCATTERING SPECTROSCOPY

The problem of protein aggregation is widely studied across a number of disciplines, where understanding the behaviour of the protein monomer, and its behaviour with co-solutes is imperative in order to devise solutions to the problem. Here we present a method for measuring the kinetics of protein aggregation based on ultra violet light scattering spectroscopy (UVLSS) across a range of NaCl conditions. Through measurement of wavelength dependant scattering and using the model protein {beta}-lactoglobulin, it is possible to isolate the thermodynamic contributions to thermal unfolding. We show that increasing NaCl concentration decreases the free energy of unfolding which is dominated by the decrease of the enthalpy contribution. This contribution is significantly larger than the decrease in change in entropy observed at higher salt concentrations between the folded and unfolded states.

biophysics↗

bioRxiv2018

Fast and Flexible GPU Accelerated Binding Free Energy Calculations within the AMBER Molecular Dynamics Package

Alchemical free energy calculations (AFE) based on molecular dynamics (MD) simulations are key tools in both improving our understanding of a wide variety of biological processes and accelerating the design and optimization of therapeutics for numerous diseases. Computing power and theory have, however, long been insufficient to enable AFE calculations to be routinely applied in early stage drug discovery. One of the major difficulties in performing AFE calculations is the length of time required for calculations to converge to an ensemble average. CPU implementations of MD based free energy algorithms can effectively only reach tens of nanoseconds per day for systems on the order of 50,000 atoms, even running on massively parallel supercomputers. Therefore, converged free energy calculations on large numbers of potential lead compounds are often untenable, preventing researchers from gaining crucial insight into molecular recognition, potential druggability, and other crucial areas of interest. Graphics Processing Units (GPUs) can help address this. We present here a seamless GPU implementation, within the PMEMD module of the AMBER molecular dynamics package, of thermodynamic integration (TI) capable of reaching speeds of >140 ns/day for a 44,907-atom system, with accuracy equivalent to the existing CPU implementation in AMBER. The implementation described here is currently part of the AMBER 18 beta code and will be an integral part of the upcoming version 18 release of AMBER.

biophysics↗

bioRxiv2018

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon - SecYMKEG - resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Forster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

biophysics↗

bioRxiv2018

Caenorhabditis elegans locomotion dynamics is affected by internally localized magnetic fields

C. elegans nematodes are a model organism used broadly to investigate the impact of environmental factors on physiology and behavior. Here, C. elegans with internalized paramagnetic nanoparticles were placed inside magnetic field to explore effects on locomotion. We hypothesize that internalized paramagnetic nanoparticles combined with external magnetic field affect C. elegans locomotion machinery. To test our hypothesis, we used young adult C. elegans fed on bacteria mixed with paramagnetic nanoparticles of 1 m, 100 nm and 40 nm diameter. The presence of nanoparticles inside the worms body (alimentary canal, body muscle) was verified by fluorescent and electron microscopy. A custom-made software was used to track freely moving C. elegans in the absence or presence of magnetic field sequentially for 200+200 sec. We used established metrics to quantify locomotion-related parameters, including posture, motion and path features. Key features of C. elegans locomotion (increased body bends and stay ratio, decreased range, forward movement, and speed along the magnetic field) were affected in worms with internalized nanoparticles of 100 nm and 1 m in the presence of magnetic field, in contrast to untreated worms. Our work contributes on clarifying the effect of internalized paramagnetic nanoparticles, combined with magnetic field, on C. elegans locomotion. Summary StatementC. elegans with internalized paramagnetic nanoparticles are placed inside magnetic field to explore effects on locomotion. Results support the potential of C. elegans to investigate the impact of the above environmental factors on behavior.

biophysics↗

bioRxiv2018

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW/cm2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined in a novel way to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux MoNaLISA enables prolonged (40-50 frames) and large (50 x 50 m2) recordings at 0.3-1.3 Hz with enhanced optical sectioning ability. We demonstrated the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

biophysics↗

bioRxiv2018

Substrate deformations induce directed keratinocyte migration

Cell migration is an essential part of many (patho)physiological processes in the body, including keratinocyte re-epithelialization of healing wounds. Recent interest in the mechanobiology of tissues suggests that physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected primary human keratinocyte behavior and that mechanical deformations in soft (~1.2 kPA) PA gels produced by neighboring cells appeared to influence the process of de novo epithelial sheet formation. In order to clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighboring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632, a rho kinase inhibitor, or blebbistatin, a non-muscle myosin II inhibitor, on the response of the cells to PA gel deformations. The results of this study indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate displacements imposed by neighboring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during normal and pathologic wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to the observed process of substrate deformation-directed keratinocyte migration.

biophysics↗

bioRxiv2018

The Principle of Similitude in Biology: From Allometry to the Formulation of Dimensionally Homogenous ‘Laws’

Meaningful laws of nature must be independent of the units employed to measure the variables. The principle of similitude (Rayleigh 1915) or dimensional homogeneity, states that only commensurable quantities (ones having the same dimension) may be compared, therefore, meaningful laws of nature must be homogeneous equations in their various units of measurement, a result which was formalized in the {Pi} theorem (Vaschy 1892; Buckingham 1914). However, most relations in allometry do not satisfy this basic requirement, including the 3/4 Law (Kleiber 1932) that relates the basal metabolic rate and body mass, besides it is sometimes claimed to be the most fundamental biological rate (Brown et al. 2004) and the closest to a law in life sciences (Brown et al. 2004). Using the {Pi} theorem, here we show that it is possible to construct an unique homogeneous equation for the metabolic rates, in agreement with data in the literature. We find that the variations in the dependence of the metabolic rates on body mass are secondary, coming from variations in the allometric dependence of the heart frequencies. This includes not only different classes of animals (mammals, birds, invertebrates) but also different aerobic conditions (basal and maximal). Our results demonstrate that most of the differences found in the allometric exponents (White et al. 2007) are due to compare incommensurable quantities and that our dimensionally homogenous formula, unify these differences into a single formulation. We discuss the ecological implications of this new formulation in the context of the Malthusians, Fenchels and Calders relations.

biophysics↗

bioRxiv2018

Dynein catch bond as a mediator of codependent bidirectional cellular transport

Intracellular bidirectional transport of cargo on Microtubule filaments is achieved by the collective action of oppositely directed dynein and kinesin motors. Experiments have found that in certain cases, inhibiting the activity of one type of motor results in an overall decline in the motility of the cellular cargo in both directions. This counter-intuitive observation, referred to as paradox of codependence is inconsistent with the existing paradigm of a mechanistic tug-of-war between oppositely directed motors. Unlike kinesin, dynein motors exhibit catchbonding, wherein the unbinding rates of these motors decrease with increasing force on them. Incorporating this catchbonding behavior of dynein in a theoretical model, we show that the functional divergence of the two motors species manifests itself as an internal regulatory mechanism and provides a plausible resolution of the paradox of codependence.

biophysics↗