SEARCH · Search bioRxiv

Results for “Biophysics”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,225 records · Page 68Linked to original sources

bioRxiv2018

DNA sequence context controls the binding and processivity of T7 DNA primase

Primases are key enzymes involved in DNA replication. They act on single-stranded DNA, and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA-binding and activity of the bacteriophage T7 primase using a new workflow called High-Throughput Primase Profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such as G/T-rich flanks, which increase primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases.

biophysics↗

bioRxiv2018

DeepLoco: Fast 3D Localization Microscopy Using Neural Networks

Single-molecule localization super-resolution microscopy (SMLM) techniques like STORM and PALM have transformed cellular microscopy by substantially increasing spatial resolution. In this paper we introduce a new algorithm for a critical part of the SMLM process: estimating the number and locations of the fluorophores in a single frame. Our algorithm can analyze a 20000-frame experimental 3D SMLM dataset in about one second -- substantially faster than real-time and existing algorithms. Our approach is straightforward but very different from existing algorithms: we train a neural network to minimize the Bayes risk under a generative model for single SMLM frames. The neural network maps a frame directly to a collection of fluorophore locations, which we compare to the ground truth using a novel loss function. While training the neural network takes several hours, it only has to be done once for a given experimental setup. After training, localizing fluorophores in new images is extremely fast -- orders of magnitude faster than existing algorithms. Faster recovery opens the door to real-time calibration and accelerated acquisition, and future work could tackle more complicated optical systems and more realistic simulators.

biophysics↗

bioRxiv2018

Collective motion conceals fitness differences in crowded cellular populations

Many cellular populations are tightly-packed, for example microbial colonies and biofilms [39, 10, 41], or tissues and tumors in multi-cellular organisms [11, 29]. Movement of one cell inside such crowded assemblages requires movement of others, so that cell displacements are correlated over many cell diameters [28, 6, 31]. Whenever movement is important for survival or growth [15, 34, 38, 9], such correlated rearrangements could couple the evolutionary fate of different lineages. Yet, little is known about the interplay between mechanical stresses and evolution in dense cellular populations. Here, by tracking deleterious mutations at the expanding edge of yeast colonies, we show that crowding-induced collective motion prevents costly mutations from being weeded out rapidly. Joint pushing by neighboring cells generates correlated movements that suppress the differential displacements required for selection to act. Such mechanical screening of fitness differences allows the mutants to leave more descendants than expected under non-mechanical models, thereby increasing their chance for evolutionary rescue [2, 5]. Our work suggests that mechanical interactions generally influence evolutionary outcomes in crowded cellular populations, which has to be considered when modeling drug resistance or cancer evolution [1, 22, 34, 30, 36, 42].

biophysics↗

bioRxiv2018

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the {beta}{gamma} subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 [A] or better. Comparison with the 3.4 [A] resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the {beta} subunit of the G protein was observed.

biophysics↗

bioRxiv2018

Spatial moment description of birth-death-movementprocesses incorporating the effects of crowding andobstacles

Birth-death-movement processes, modulated by interactions between individuals, are fundamental to many cell biology processes. A key feature of the movement of cells within in vivo environments are the interactions between motile cells and stationary obstacles. Here we propose a multi-species model of individual-level motility, proliferation and death. This model is a spatial birth-death-movement stochastic process, a class of individual-based model (IBM) that is amenable to mathematical analysis. We present the IBM in a general multi-species framework, and then focus on the case of a population of motile, proliferative agents in an environment populated by stationary, non-proliferative obstacles. To analyse the IBM, we derive a system of spatial moment equations governing the evolution of the density of agents and the density of pairs of agents. This approach avoids making the usual mean-field assumption so that our models can be used to study the formation of spatial structure, such as clustering and aggregation, and to understand how spatial structure influences population-level outcomes. Overall the spatial moment model provides a reasonably accurate prediction of the system dynamics, including important effects such as how varying the properties of the obstacles leads to different spatial patterns in the population of agents.

biophysics↗

bioRxiv2018

High precision wavefront control in Point Spread Function engineering for single emitter localization

Point spread function (PSF) engineering is used in single emitter localization to measure the emitter position in 3D and possibly other parameters such as the emission color or dipole orientation as well. Advanced PSF models such as spline fits to experimental PSFs or the vectorial PSF model can be used in the corresponding localization algorithms in order to model the intricate spot shape and deformations correctly. The complexity of the optical architecture and fit model makes PSF engineering approaches particularly sensitive to optical aberrations. Here, we present a calibration and alignment protocol for fluorescence microscopes equipped with a spatial light modulator (SLM) with the goal of establishing a wavefront error well below the diffraction limit for optimum application of complex engineered PSFs. We achieve high-precision wavefront control, to a level below 20 m{lambda} wavefront aberration over a 30 minute time window after the calibration procedure, using a separate light path for calibrating the pixel-to-pixel variations of the SLM, and alignment of the SLM with respect to the optical axis and Fourier plane within 3 {micro}m (x/y) and 100 {micro}m (z) error. Aberrations are retrieved from a fit of the vectorial PSF model to a bead z-stack and compensated with a residual wavefront error comparable to the error of the SLM calibration step. This well-calibrated and corrected setup makes it possible to create complex 3D+{lambda} PSFs that fit very well to the vectorial PSF model. Proof-of-principle bead experiments show precisions below 10 nm in x, y, and{lambda} , and below 20 nm in z over an axial range of 1 {micro}m with 2000 signal photons and 12 background photons.

biophysics↗

bioRxiv2018

Live single molecule microscopy of HIV-1 assembly in host T cells reveals a spatio-temporal effect of the viral genome.

Monitoring virus assembly dynamic at the nanoscale level in host cells remains a major challenge. Human Immunodeficiency Virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100nm in diameter. HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble immature virus. Here, we monitored Gag assembly in host CD4 T lymphocytes using single molecule dynamics microscopy and energy mapping. A workflow allowing long time recordings of single Gag molecule localization, diffusion and effective energy maps was developed for robust quantitative analysis of HIV assembly and budding. Comparison of numerous cell plasma membrane assembling platforms in cells expressing wild type or assembly-defective Gag proteins showed that VLP formation last 15 minutes, with an assembly time of 5 minutes, and that the nucleocapsid domain is mandatory. Importantly, it reveals that the viral genome coordinates spatio-temporally HIV-1 assembly.

biophysics↗

bioRxiv2018

Efflux Pumps Represent Possible Evolutionary Convergence onto the Beta Barrel Fold

There are around 100 types of integral outer membrane proteins in each Gram negative bacteria. All of these proteins have the same fold--an up-down {beta}-barrel. It has been suggested that all membrane {beta}-barrels other than lysins are homologous. Here we suggest that {beta}-barrels of efflux pumps have converged on this fold as well. By grouping structurally-solved outer membrane {beta}-barrels (OMBBs) by sequence we find evidence that the membrane environment may have led to convergent evolution of the barrel fold. Specifically, the lack of sequence linkage to other barrels coupled with distinctive structural differences, such as differences in strand tilt and barrel radius, suggest that efflux pumps have evolutionarily converged on the barrel. Finally, we find a possible ancestor for the OMBB efflux pumps as they are related to periplasmic components of the same pumps.

biophysics↗

bioRxiv2018

Identification and Structure-Activity Relationship of HDAC6 Zinc-finger Ubiquitin Binding Domain Inhibitors

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation, and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of a HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155 are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens-up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

biophysics↗

bioRxiv2018

Dissecting Conformational Changes in APP’s Transmembrane Domain Linked to Altered ε-Efficiency of Familial Alzheimer’s Disease Mutants

The mechanism by which familial Alzheimers disease (FAD) mutations within the transmembrane domain (TMD) of the Amyloid Precursor Protein (APP) affect [a]-endoproteolysis is only poorly understood. Thereby, mutations in the cleavage domain reduce [a]-efficiency of a-secretase cleavage and some even shift entry into production lines. Since cleavage occurs within the TMD, a relationship between processing and TMD structure and dynamics seems obvious. Using molecular dynamic simulations, we dissect the dynamic features of wild type and seven FAD-mutants into local and global components. Mutations consistently enhance hydrogen8 bond fluctuations upstream of the [a]-cleavage sites but maintain strong helicity there. Dynamic perturbation response scanning reveals that FAD-mutants target backbone motions utilized in the bound state. Those motions, obscured by large-scale motions in the pre-bound state, provide (i) a dynamic mechanism underlying the proposedcoupling between binding and [a]-cleavage, (ii) key sites consistent with experimentally determined docking sites, and (iii) the distinction between mutants and wild-type.

biophysics↗

bioRxiv2018

Information transfer in QT-RR dynamics: Towards a model-free QT correction method

The relation between the electrical properties of the heart and the beating rate is essential for the heart functioning. This relation is central when calculating the \"corrected QT interval\" -- an important measure of the risk of potentially lethal arrhythmias. We use the transfer entropy method from information theory to quantitatively study the mutual dynamics of the ventricular action potential duration (the QT interval) and the length of the beat-to-beat (RR) interval. We show that for healthy individuals there is a strong asymmetry in the information transfer: the information flow from RR to QT dominates over the opposite flow (from QT to RR), i.e. QT depends on RR to a larger extent than RR on QT. Moreover, the history of the intervals has a strong effect on the information transfer: at sufficiently long QT history length the information flow asymmetry inverts and the RR influence on QT dynamics weakens. Finally, we demonstrate that the widely used QT correction methods cannot properly capture the changes in the information flows between QT and RR. We conclude that our results obtained through a model-free informational perspective can be utilised to improve and test the QT correction schemes in clinics.

biophysics↗

bioRxiv2018

Differential Recordings of Local Field Potential: A Genuine Tool to Quantify Functional Connectivity

Local field potential (LFP) recording is a very useful electrophysiological method to study brain processes. However, this method is criticized for recording low frequency activity in a large area of extracellular space potentially contaminated by distal activity. Here, we theoretically and experimentally compare ground-referenced (RR) with differential recordings (DR). We analyze electrical activity in the rat cortex with these two methods. Compared with RR, DR reveals the importance of local phasic oscillatory activities and their coherence between cortical areas. Finally, we show that DR provides a more faithful assessment of functional connectivity caused by an increase in the signal to noise ratio, and of the delay in the propagation of information between two cortical structures.

biophysics↗

bioRxiv2018

Structure of WT1 zinc fingers bound to its cognate DNA: Implications of the KTS insert

WT1 is a transcription factor with a DNA binding N-terminal domain containing four C2H2-type zinc fingers. In order to perform its role as a transcription factor, WT1 needs to specifically recognize and properly bind to its target DNA. How this is done is still not completely clear. Two of WT1s major isoforms are distinguished by the presence or absence of a 3 amino acid insert, Lysine-Threonine-Serine (KTS) in the linker between zinc-fingers 3 and 4. This KTS insert is conserved throughout all life forms expressing WT1. The -KTS isoform, which acts as a transcription factor, binds DNA with higher affinity than the +KTS isoform, which is thought to participate in RNA splicing and interaction with partner proteins. This study was aims at elucidating the effect of the KTS insert on DNA binding. Here we present the crystal structure of WT1 zinc fingers 2-4, with and without the KTS insert, bound to the WT1 9-base pair cognate DNA sequence, refined to 1.9 [A] and 2.5 [A] respectively. The structures show that the +KTS isoform of WT1 recognizes DNA with the same specificity as the -KTS isoform. The only differences in the DNA bound conformation of the two isoforms are found within the linker containing the KTS, and these mainly involve the loss of the C-capping interactions thought to stabilize the complex. These structures provide the molecular detail necessary for the interpretation of the WT1 transcriptional DNA recognition and validation of its transcriptional targets.

biophysics↗

bioRxiv2018

Hamiltonian distributed chaos in DNA

It is shown that distributed chaos, generated by Hamiltonian DNA dynamics with spontaneously broken time translational symmetry, imprints itself on the DNA sequence of Arabidopsis thaliana (a model plant for genetic sequencing and mapping) and of the NRXN1 and BRCA2 human genes (as an example). The base-stacking interactions in the DNA duplex, degenerate codon groups and a relation to the KAM theory have been discussed in this context.

biophysics↗

bioRxiv2018

Mitochondrial ATP synthase dimers spontaneously self-associate driven by a long-ranged membrane-induced force

ATP synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. Cryo-electron tomograms of these membranes from yeast to vertebrates have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. These rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. We present the result of a quantitative molecular-simulation analysis that strongly suggests that the ATP synthase dimers organize into rows spontaneously, driven by a long-ranged attractive force that results from relief in the overall elastic strain of the membrane. This strain is caused by the V-like shape of the dimers, unique among membrane-protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of protein-protein interactions, or of a specific lipid composition of the membrane. We further hypothesize that once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations, by causing macroscopic membrane ridges that ultimately become the cristae edges. In this view, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline the key experiments that would be required to verify or refute this hypothesis.

biophysics↗

bioRxiv2018

How well do force fields capture the strength of salt bridges in proteins?

Salt bridges form between pairs of ionisable residues in close proximity and are important interactions in proteins. While salt bridges are known to be important both for protein stability, recognition and regulation, we still do not have fully accurate predictive models to assess the energetic contributions of salt bridges. Molecular dynamics simulations is one technique that may be used study the complex relationship between structure, solvation and energetics of salt bridges, but the accuracy of such simulations depend on the force field used. We have used NMR data on the B1 domain of protein G (GB1) to benchmark molecular dynamics simulations. Using enhanced sampling simulations, we calculated the free energy of forming a salt bridge for three possible ionic interactions in GB1. The NMR experiments showed that these interactions are either not formed, or only very weakly formed, in solution. In contrast, we show that the stability of the salt bridges is slightly overestimated in simulations of GB1 using six commonly used combinations of force fields and water models. We therefore conclude that further work is needed to refine our ability to model quantitatively the stability of salt bridges through simulations, and that comparisons between experiments and simulations will play a crucial role in furthering our understanding of this important interaction.

biophysics↗

bioRxiv2018

Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy

When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labeling or there is concern that labelling will disrupt its function. Commonly used techniques for observing unlabeled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments, and interferometric scattering has a limited field of view. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the image quality of interference reflection microscopy is similar to or exceeds that of all other techniques and, with minimal microscope modification, can be used to study the dynamics of unlabeled microtubules.

biophysics↗

bioRxiv2018

Compartmentalized RNA catalysis in membrane - free coacervate protocells

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalization via coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

biophysics↗