ALBERT

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Daniel Trotter, Stefan Wallin〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Experiments have compared the folding of proteins with different amino acid sequences but the same basic structure, or fold. Results indicate that folding is robust to sequence variations for proteins with some nonlocal folds, such as all-〈em〉β〈/em〉, while the folding of more local, all-〈em〉α〈/em〉 proteins typically exhibit a stronger sequence dependence. Here we use a coarse-grained model to systematically study how variations in sequence perturb the folding energy landscapes of three model sequences with 3〈em〉α〈/em〉, 4〈sup〉β+α〈/sup〉 and 〈em〉β〈/em〉-barrel folds, respectively. These three proteins exhibit folding features in line with experiments, including expected rank order in the cooperativity of the folding transition and stability-dependent shifts in the location of the free energy barrier to folding. Using a generalized-ensemble simulation approach, we determine the thermodynamics of around 2,000 sequence variants representing all possible hydrophobic/polar single- and double-point mutations. From an analysis of the subset of stability neutral mutations we find that folding is perturbed in a topology-dependent manner, with the 〈em〉β〈/em〉-barrel protein being the most robust. Our analysis shows, in particular, that the magnitude of mutational perturbations of the transition state is controlled in part by the size or “width” of the underlying conformational ensemble. This result suggests that the mutational robustness of the folding of the 〈em〉β〈/em〉-barrel protein is underpinned by its conformationally restricted transition state ensemble, revealing a link between sequence and topological effects in protein folding.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Reut Israeli, Sofiya Kolusheva, Uzi Hadad, Raz Jelinek〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Membrane interactions of amyloidogenic proteins constitute central determinants both in protein aggregation as well as amyloid cytotoxicity. Most reported studies of amyloid peptide-membrane interactions have employed model membrane systems, combined with application of spectroscopy methods or microscopy analysis of individual binding events. Here, we applied for the first time imaging flow cytometry for investigating interactions of representative amyloidogenic peptides – the 102-126 fragment of prion protein [PrP(106-126)], and the human Islet Amyloid Polypeptide (hIAPP) – with giant lipid vesicles. Imaging flow cytometry was also applied to examine the inhibition of PrP(106-126)-membrane interactions by epigallocatechin gallate (EGCG), a known modulator of amyloid peptide aggregation. We show that imaging flow cytometry provided comprehensive population-based statistical information upon morphology changes of the vesicles induced by PrP(106-126) and hIAPP. Specifically, the experiments reveal that both PrP(106-126) and hIAPP induced dramatic transformations of the vesicles, specifically disruption of the spherical shapes, reduction of vesicle circularity, lobe formation, and modulation of vesicle compactness. Interesting differences, however, were apparent between the impact of the two peptides upon the model membranes. The morphology analysis also showed that EGCG ameliorated vesicle disruption by PrP(106-126). Overall, this study demonstrates that imaging flow cytometry provides powerful means for disclosing population-based morphological membrane transformations induced by amyloidogenic peptides and their inhibition by aggregation modulators.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Wajih Anwer, Amanda Ratto Velasquez, Valeria Tsoukanova〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Excessive accumulation of acylcarnitines (ACs), often caused by metabolic disorders, has been associated with obesity, arrhythmias, cardiac ischemia, insulin resistance, etc. Mechanisms whereby elevated ACs might contribute to pathophysiological effects remain largely unexplored. We have aimed to gain insight into AC interactions with the mitochondrial inner membrane. To model its outer leaflet, Langmuir monolayers and cushioned supported bilayers were employed. Their interactions with ACs were monitored with epifluorescence microscopy, which revealed a local leaflet expansion upon exposure to elevated concentrations of a long-chain AC, plausibly caused by its insertion. To assess the AC insertion parameters, constant pressure insertion assays were performed. A value of 21 ± 3 Å〈sup〉2〈/sup〉 was obtained for the AC insertion area, which is roughly the same as the cross-sectional area of an acyl chain. By contrast, the carnitine moiety was found to require an area of 37 ± 3 Å〈sup〉2〈/sup〉. The AC insertion has thus been concluded to involve solely the AC acyl chain. This mode of insertion implies that the carnitine moiety with its nontitratable positive charge is left dangling at the membrane surface, which is likely to alter the surface electrostatics of the outer leaflet. The extrapolation of these findings has enabled us to hypothesize that, by altering the morphology and surface electrostatics of the outer leaflet, the insertion of ACs, in particular their long-chain counterparts, may trigger a nonspecific activation of signaling pathways in the inner mitochondrial membrane thereby modulating its function and potentially leading to pathophysiological responses.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Chris Neale, Angel E. García〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Mutant Ras proteins are important drivers of human cancers, yet no approved drugs act directly on this difficult target. Over the last decade, the idea has emerged that oncogenic signaling can be diminished by molecules that drive Ras into orientations in which effector binding interfaces are occluded by the cell membrane. To support this approach to drug discovery, we characterize the orientational preferences of membrane-bound K-Ras4B in 1.45 milliseconds aggregate time of atomistic molecular dynamics simulations. Individual simulations probe active or inactive states of Ras on membranes with or without anionic lipids. We find that the membrane orientation of Ras is relatively insensitive to its bound guanine nucleotide and activation state but depends strongly on interactions with anionic phosphatidylserine lipids. These lipids slow Ras’ translational and orientational diffusion and promote a discrete population in which small changes in orientation control Ras’ competence to bind multiple regulator and effector proteins. Our results suggest that compound-directed conversion of constitutively active mutant Ras into functionally inactive forms may be accessible via subtle perturbations of Ras’ orientational preferences at the membrane surface.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 23 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Jun Wang, Chitra Karki, Yi Xiao, Lin Li〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Ribosomes are essential machines for protein synthesis in cells. Their structures are very complex but conserved in different species. Since most parts of a ribosome are composed of negatively charged RNAs, its electrostatics should play a fundamental role in the realization of its functions. However, a complete picture of the electrostatics of ribosomes is still absent at present. Here, assisted by the latest version of DelPhi (Version: 8.4), we illustrate a picture of the electrostatics of a prokaryotic ribosome as well as its molecular chaperones. The revealed electrostatics features are well consistent with available experimental data as well as the functions of the ribosome and its molecular chaperones and provides a basis for further studying the mechanism underlying these functions.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 23 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): T.B. Brouwer, N. Hermans, J. van Noort〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables (LUT). Here, we introduce an alternative 3D phasor algorithm, that provides robust bead tracking with nanometric localization accuracy in a 〈math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.svg"〉〈mi〉z〈/mi〉〈/math〉〈em〉-〈/em〉range of over 10 μm under non-optimal imaging conditions. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. The accuracy of 3D Phasor tracking was extensively tested and compared to a LUT approach using Lorentz Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required and image artefacts are difficult to avoid.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Francisco Sahli-Costabal, Kinya Seo, Euan Ashley, Ellen Kuhl〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉All medications have adverse effects. Among the most serious of these are cardiac arrhythmias. Current paradigms for drug safety evaluation are costly, lengthy, conservative, and impede efficient drug development. Here we combine multiscale experiment and simulation, high-performance computing, and machine learning to create a risk estimator to stratify new and existing drugs according to their pro-arrhythmic potential. We capitalize on recent developments in machine learning and integrate information across ten orders of magnitude in space and time to provide a holistic picture of the effects of drugs, either individually or in combination with other drugs. We show, both experimentally and computationally, that drug-induced arrhythmias are dominated by the interplay between two currents with opposing effects: the rapid delayed rectifier potassium current and the L-type calcium current. Using Gaussian process classification, we create a classifier that stratifies drugs into safe and arrhythmic domains for any combinations of these two currents. We demonstrate that our classifier correctly identifies the risk categories of 22 common drugs, exclusively on the basis of their concentrations at 50% current block. Our new risk assessment tool explains under which conditions blocking the L-type calcium current can delay or even entirely suppress arrhythmogenic events. Using machine learning in drug safety evaluation can provide a more accurate and comprehensive mechanistic assessment of the pro-arrhythmic potential of new drugs. Our study paves the way towards establishing science-based criteria to accelerate drug development, design safer drugs, and reduce heart rhythm disorders.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Alemayehu A. Gorfe, Stephen G. Sligar〈/p〉

Description: 〈p〉Publication date: Available online 22 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Christopher T. Lee, Justin G. Laughlin, John B. Moody, Rommie E. Amaro, J. Andrew McCammon, Michael J. Holst, Padmini Rangamani〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Advances in imaging methods such as electron microscopy, tomography, and other modalities are enabling high-resolution reconstructions of cellular and organelle geometries. Such advances pave the way for using these geometries for biophysical and mathematical modeling once these data can be represented as a geometric mesh, which, when carefully conditioned, enables the discretization and solution of partial differential equations. In this study, we outline the steps for a naïve user to approach 〈code〉GAMer 2〈/code〉, a mesh generation code written in C++ designed to convert structural datasets to realistic geometric meshes, while preserving the underlying shapes. We present two example cases, 1) mesh generation at the subcellular scale as informed by electron tomography, and 2) meshing a protein with structure from x-ray crystallography. We further demonstrate that the meshes generated by 〈code〉GAMer〈/code〉 are suitable for use with numerical methods. Together, this collection of libraries and tools simplifies the process of constructing realistic geometric meshes from structural biology data.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Brian P. Josey, Frank Heinrich, Vitalii Silin, Mathias Lösche〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Aimed to reproduce the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neurotransmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neurotransmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflection measurements on artificial membranes, so-called sparsely-tethered bilayer lipid membranes (stBLMs), reveal the structural aspects of neurotransmitters association with zwitterionic and anionic bilayers. We thus establish that serotonin interacts non-specifically with the membrane at physiologically relevant concentrations whilst GABA (γ-aminobutyric acid) does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity and neutron reflectometry shows that it penetrates the membrane deeply whereas GABA is excluded from the bilayer.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Amulya Nidhi Shrivastava, Luc Bousset, Marianne Renner, Virginie Redeker, Jimmy Savistchenko, Antoine Triller, Ronald Melki〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The aggregation of the protein α-Synuclein (α-Syn) leads to different synucleinopathies. We recently showed that structurally distinct fibrillar α-Synuclein polymorphs trigger either Parkinson’s Disease or Multiple System Atrophy hallmarks 〈em〉in vivo〈/em〉. Here, we establish structural-molecular basis for these observations. We show that distinct fibrillar α-Syn polymorphs bind to and cluster differentially at the plasma membrane in both primary neuronal cultures and organotypic hippocampal slice cultures from wild-type mice. We demonstrate a polymorph-dependent and concentration-dependent seeding. We show a polymorph-dependent differential synaptic re-distribution of α3-Na〈sup〉+〈/sup〉/K〈sup〉+〈/sup〉-ATPase, GluA2-AMPA and GluN2B-NMDA receptors but not GluA1-AMPA and mGluR5 receptors. We also demonstrate polymorph-dependent alteration in neuronal network activity upon seeded aggregation of α-Syn. Our findings bring new insight into how distinct α-Syn polymorphs differentially bind to and seed monomeric α-Syn aggregation within neurons, thus affecting neuronal homeostasis through the redistribution of synaptic proteins.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Alena Klindziuk, Billie Meadowcroft, Anatoly B. Kolomeisky〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Populations of genetically identical cells generally show a large variability in cell phenotypes, which is typically associated with the stochastic nature of gene expression processes. It is widely believed that a significant source of such randomness is the transcriptional bursting, which is when periods of active production of RNA molecules alternate with periods of RNA degradation. However, the molecular mechanisms of such strong fluctuations remain unclear. Recent studies suggest that DNA supercoiling, which happens during transcription, might be directly related to the bursting behavior. Stimulated by these observations, we developed a stochastic mechano-chemical model of supercoiling-induced transcriptional bursting where the RNA synthesis leads to the buildup of torsion in DNA. This slows down the RNA production until binding of an enzyme gyrase to DNA, which releases the stress and allows for the RNA synthesis to restart with the original rate. Using a thermodynamically consistent coupling between mechanical and chemical processes, dynamic properties of transcription are explicitly evaluated. In addition, a first-passage method to evaluate the dynamics of transcription is developed. Theoretical analysis shows that the transcriptional bursting is observed when both the supercoiling and the mechanical stress-release due to gyrase are present in the system. It is also found that the overall RNA production rate is not constant and depends on the number of previously synthesized RNA molecules. A comparison with experimental data on bacteria allows us to evaluate the energetic cost of supercoiling during transcription. It is argued that the relatively weak mechanochemical coupling might allow transcription to be regulated most effectively.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 21 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): M.R. McCarthy, Y. Savich, R.L. Cornea, D.D. Thomas〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N- and C-) with fluorescent probes, and used intramolecular TR-FRET to assess inter-lobe distances when CaM is bound to either RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an inter-lobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca〈sup〉2+〈/sup〉, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca〈sup〉2+〈/sup〉 the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca〈sup〉2+〈/sup〉, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: (1) The two conformations are resolved and more ordered. (2) The open state is the major component. (3) Ca〈sup〉2+〈/sup〉 stabilized the closed conformation by a factor of two. We conclude that the Ca〈sup〉2+〈/sup〉-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca〈sup〉2+〈/sup〉-dependent structural dynamics of bound CaM.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 16 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Jan Huertas, Caitlin M. MacCarthy, Hans R. Schöler, Vlad Cojocaru〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence specific manner. Here we reveal the structural dynamics of nucleosomes that mediate Oct4 binding from molecular dynamics simulations. Nucleosome flexibility and the amplitude of nucleosome motions such as breathing and twisting are enhanced in nucleosomes with multiple TF binding sites. Moreover, the regions around the binding sites display higher local structural flexibility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations in which Oct4 recognizes partial binding sites display stable TF-DNA interactions similar to those observed in complexes with free DNA and compatible with the DNA curvature and DNA-histone interactions. Therefore, we propose a structural basis for nucleosome recognition by a pioneer TF which is essential for understanding how chromatin is unraveled during cell fate conversions.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 16 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): P. Garidel, A. Eiperle, M. Blech, J. Seelig〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pre-transition at 72 °C with an unfolding enthalpy of ΔH〈sub〉cal〈/sub〉 ∼ 200-300 kcal/mol and a main transition at 85 °C with an enthalpy of ∼900 - 1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analysed with the multi-state Zimm-Bragg theory. For the investigated mAb, unfolding is characterised by a cooperativity parameter σ ∼6x10〈sup〉−5〈/sup〉 and a Gibbs free energy of unfolding of g〈sub〉nu〈/sub〉 ∼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, ν∼220±50 amino acids are involved in the pre-transition and ν∼850±30 in the main transition, accounting for ∼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔH〈sub〉cal〈/sub〉 and lowers the midpoint temperature T〈sub〉m〈/sub〉. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25 °C are predicted to be 2-3 M for the pre-transition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCL molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pre-transition and 0.55 for the main transition. The pre-transition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 18 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Yuka Sakuma, Toshihiro Kawakatsu, Takashi Taniguchi, Masayuki Imai〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉In cell membranes, the functional constituents, such as peptides, proteins, and polysaccharides, diffuse in a sea of lipids as single molecules and molecular aggregates. Thus, the fluidity of the heterogeneous multicomponent membrane is important to understand the roles of the membrane in cell functionality. Recently, Henle and Levine described the hydrodynamics of molecular diffusion in a spherical membrane. A tangential point force at the north pole induces a pair of vortices whose centers lie on a line perpendicular to the point force and are symmetrical with respect to the point force. The position of the vortex center depends on ηm /Rηw where R is the radius of the spherical membrane, and ηm and ηw are the viscosities of the membrane and the surrounding medium, respectively. Based on this theoretical prediction, we applied a point force to a phase separated spherical vesicle composed of DPPC/DOPC/Cholesterol by means of a microinjection technique. The path lines were visualized by trajectories of microdomains. We determined the position of the vortex center and estimated the membrane viscosity using the dependence of the position of the vortex center on ηm/Rηw. The obtained apparent membrane viscosities for various compositions are mapped on the phase diagram. The membrane viscosity is almost constant in the range of 0 〈 ϕLo ≤ 0.5 (ϕLo: area fraction of the liquid ordered phase), whereas that in the range of 0.5 ≤ ϕLo 〈 1.0 exponentially increases with increasing of ϕLo. The obtained viscosity landscape provides a basic understanding of the fluidity of heterogeneous multicomponent membranes.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 17 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): L. Lanzanò〈/p〉

Description: 〈p〉Publication date: Available online 15 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): C. Ashley Barnes, Yang Shen, Jinfa Ying, Ad Bax〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated by NMR spectroscopy the effect of substituting key charged Arg, Lys, Glu and Asp residues by Gly or His. Results indicate that such mutations result in transient breaking of the helix at the site of mutation, but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH helical stability. Whereas a single Gly substitution caused transient breaks for 〈em〉ca〈/em〉 20% of the time, two consecutive Gly substitutions break the helix for 〈em〉ca〈/em〉 65% of the time. NMR relaxation measurements indicate that the exchange rate between intact and broken helix is fast (〉300,000 s〈sup〉-1〈/sup〉) and that for the wild-type sequence the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, and not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, where the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 14 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Shawn M. Sternisha, A. Carl Whittington, Juliana A. Martinez Fiesco, Carol Porter, Malcolm M. McCray, Timothy Logan, Cristina Olivieri, Gianluigi Veglia, Peter J. Steinbach, Brian G. Miller〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence and 〈sup〉19〈/sup〉F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme’s intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, non-cooperative variants.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 31 December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Anisha Shakya, Seonyoung Park, Neha Rana, John T. King〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1〈em〉α〈/em〉), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1〈em〉α〈/em〉 is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 25 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Mauro L. Mugnai, Matthew A. Caporizzo, Yale E. Goldman, D. Thirumalai〈/p〉

Description: 〈p〉Publication date: Available online 25 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Hiroaki Yokota〈/p〉

Description: 〈p〉Publication date: Available online 18 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Hongchun Li, Pemra Doruker, Guang Hu, Ivet Bahar〈/p〉

Description: 〈p〉Publication date: Available online 15 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Benjamin W. Cook, Rachel E. Lacoursiere, Gary S. Shaw〈/p〉

Description: 〈p〉Publication date: Available online 15 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Luis F. Delgadillo, Graham A. Marsh, Richard E. Waugh〈/p〉

Description: 〈p〉Publication date: Available online 15 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Ivan Hornak, Heiko Rieger〈/p〉

Description: 〈p〉Publication date: Available online 12 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Md Shahinuzzaman, Dipak Barua〈/p〉

Description: 〈p〉Publication date: Available online 12 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Christopher M. MacDermaid, Kyle Wm. Hall, Russell H. DeVane, Michael L. Klein, Giacomo Fiorin〈/p〉

Description: 〈p〉Publication date: Available online 12 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Mary Pitman, Yamini Dalal, Garegin A. Papoian〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Taylor Jones, Bianxiao Cui〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Nicholas A. Vickers, Sean B. Andersson〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Sophia Gruber, Achim Löf, Tobias Obser, Maria A. Brehm, Martin Benoit, Jan Lipfert〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Jody A. Dantzig, Him Shweta, Yale E. Goldman〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Ehsan Akbari, Melika Shahhosseini, Jonathan W. Song, Carlos E. Castro〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Rachel A. Munro, Jeffrey De Vlugt, Vladimir Ladizhansky, Leonid S. Brown〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Jason Cote〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Marina Kirichenko, Leonid Chaikov, Svetlana Krivokhizha, Nikolay Bulychev〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Sina Jazani, Steve Pressé〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Nicholas S. McCarty, Manuel Razo-Mejia, Rob Phillips〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Chengzhi He, Yayan Xie〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Tyler Elmendorf, Martha Scharlau, Francis Millett〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Greg Morrin〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Andrew R. Mason, William D. Jamieson, Oliver Williams, Daniel Slocombe, Oliver K. Castell〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Sarika Kumari, Michael R. Morrow, Valerie Booth〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Yanitza Trosel, Valerie Booth, Anand Yethiraj〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Stephen B. Lenzini, Raymond Bargi, Gina Chung, Jae-Won Shin〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Takashi Terabayashi, Kenji Fukuda, Minoru Morita, Tadasu Urashima〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Yu-An Kuo, Oliver S. Zhao, Hung-Che Kuo, James R. Rybarski, Trung D. Nguyen, Yuan-I Chen, Soonwoo Hong, Yen-Liang Liu, Ilya J. Finkelstein, Tim Yeh〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Ernie Au〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Simon Albertini, Felix Laimer, Lukas Tiefenthaler, Sarah Flatscher, Fabio Zappa, Mariana de Araujo, Harald Schöbel, Lukas A. Huber, Paul Scheier〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): K.V. Lakshmi, Vidmantas Kalendra, Gourab Banerjee, Ipsita Ghosh, Ke Yang, Victor S. Batista, Gary W. Brudvig〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Benjamin Ambrose, Matthew Willmott, Tristan Johnston-Wood, Robert A. Shaw, J.G. Hill, Timothy D. Craggs〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Adam A. Jabak, Nicholas Bryden, Fredrik Westerlund, Per Lincoln, Micah J. McCauley, Ioulia F. Rouzina, Mark C. Williams, Thayaparan Paramanathan〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Brendan R. Deal〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Julia Eckert, Luca Giomi, Thomas Schmidt〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Atsushi Miyagi, Simon Scheuring〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Samantha M. Betts, Jazmine M. Richardson, Eldon R. Hard, John M. Franck〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Haixia Wang, Rui Ma, Karin Nienhaus, G. Ulrich Nienhaus〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Olusola Ladokun, Sarah O. Oni, Olawale Akanbi〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Joanna P. Patalas, Karolina Rucinska, Agata Szymbor, Żaneta Polańska, Michał Taube, Augustyn Molinski, Zuzanna Pietralik, Barbara Peplińska, Agnieszka Boś-Liedke, Maciej Kozak〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Xian Wang, Changhong Cao, Jingcheng Shan, Yakun Zhao, Tobin Filleter, Yu Sun〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Anne E. Murdaugh, Audrey Smith〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Chih-Yu Yang, Maja Bialecka-Fornal, Colleen Weatherwax, Joseph Larkin, Arthur Prindle, Jintao Liu, Jordi Garcia-Ojalvo, Gurol M. Suel〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Jiaxin Sun, Avinash K. Thakur, Liviu Movileanu〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Dayo D. Adeyemo, Praise Farayola, Oluwaseun Egunsola, Bernard M. Hangòrmbe〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Ye Lin, Sean B. Andersson〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Joshua A. Johnson, Abhilasha Dehankar, Carlos E. Castro, Jessica Winter〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Yuan-I Chen, Yin-Jui Chang, Trung D. Nguyen, Cong Liu, Stephanie Phillion, Yu-An Kuo, Huong T. Vu, Angela Liu, Yen-Liang Liu, Soonwoo Hong, Hsin-Chin Li, Pengyu Ren, Thomas E. Yankeelov, Tim Yeh〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Rong Zhu, Julia Gobl, Marion Holy, Oliver Kudlacek, Walter Sandtner, Thomas Stockner, Hermann J. Gruber, Michael Freissmuth, Amy Hauck Newman, Harald H. Sitte, Peter Hinterdorfer〈/p〉

Description: 〈p〉Publication date: 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 3, Supplement 1〈/p〉 〈p〉Author(s): Joel Lapin, Emmanuel Awosanya, Alexander Nevzorov〈/p〉

Description: 〈p〉Publication date: Available online 7 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Matthew C. Blosser, Aurelia R. Honerkamp-Smith〈/p〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Emilie Gachon, Patrick Mesquida〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The mechanical properties of collagen fibrils play an important role in cell-matrix interactions and are a manifestation of their molecular structure. Using a novel combination of uniaxial, longitudinal straining and radial nanoindentation, we found that type-I-collagen fibrils show a pronounced non-linear behavior in form of strain-stiffening at strains from zero to 15%, followed by strain-softening at strains from 15% to 25%. At the molecular scale, this surprising phenomenon can be explained by the combination of unfolding of disordered domains and breaking of native cross-links at different stages of strain. Fibrils cross-linked artificially by glutaraldehyde do not show such a behaviour and nanoindentation allowed us to measure the mechanics of overlap and gap region in the D-banding individually. The results could have consequences on our understanding of matrix mechanics and the influence of excessive glycation, which has been linked with age-related diseases such as diabetes. Furthermore, the simplicity of the straining method could be attractive in other areas of biophysics at the nanometer scale as it does not require any bespoke instrumentation and is easy to use.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 5 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Sean T. Vittadello, Scott W. McCue, Gency Gunasingh, Nikolas K. Haass, Matthew J. Simpson〈/p〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Sachin Katti, Sarah B. Nyenhuis, Bin Her, David S. Cafiso, Tatyana I. Igumenova〈/p〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Premila P. Samuel, Mark A. White, William C. Ou, David A. Case, George N. Phillips, John S. Olson〈/p〉

Description: 〈p〉Publication date: Available online 5 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Tadashi Nakano, Yutaka Okaie, Yasuha Kinugasa, Takako Koujin, Tatsuya Suda, Yasushi Hiraoka, Tokuko Haraguchi〈/p〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Quinn MacPherson, Bruno Beltran, Andrew J. Spakowitz〈/p〉

Description: 〈p〉Publication date: Available online 5 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Juliette T.J. Lecomte〈/p〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Cole Zmurchok, William R. Holmes〈/p〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Prasad Bandarkar, Huan Yang, Robert.Y. Henley, Meni Wanunu, Paul C. Whitford〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise and blockage currents. By manufacturing sufficiently small pores, each tRNA is required to undergo a deformation in order to translocate. Accordingly, differences in nanopore translocation times/distributions may be used to infer the mechanical properties of individual tRNA molecules. In order to bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics (MD) simulations using a simplified “structure-based” energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural/energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Taraknath Mandal, Wilson Lough, Saverio E. Spagnolie, Anjon Audhya, Qiang Cui〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The endosomal sorting complex required for transport (ESCRT) machinery carries out the membrane scission reactions that are required for many biological processes throughout cells. How ESCRTs bind and deform cellular membranes and ultimately produce vesicles has been a matter of active research in recent years. In this study, we use fully atomistic molecular dynamics simulations to scrutinize the structural details of a filament composed of Vps32 protomers, a major component of ESCRT-III complexes. The simulations show that both hydrophobic and electrostatic interactions between monomers help maintain the structural stability of the filament, which exhibits an intrinsic bend and twist. Our findings suggest that the accumulation of bending and twisting stresses as the filament elongates on the membrane surface likely contribute to the driving force for membrane invagination. The filament exposes a large cationic surface that senses the negatively charged lipids in the membrane, and the N-terminal amphipathic helix of the monomers not only acts as a membrane anchor but also generates significant positive membrane curvature. Taking all results together, we discuss a plausible mechanism for membrane invagination driven by ESCRT-III.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Alessia Centi, Arghya Dutta, Sapun H. Parekh, Tristan Bereau〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Small solutes have been shown to alter the lateral organization of cell membranes and reconstituted phospholipid bilayers; however, the mechanisms by which these changes happen are still largely unknown. Traditionally, both experiment and simulation studies have been restricted to testing only a few compounds at a time, failing to identify general molecular descriptors or chemical properties that would allow extrapolating beyond the subset of considered solutes. In this work, we probe the competing energetics of inserting a solute in different membrane environments by means of the potential of mean force. We show that these calculations can be used as a computationally-efficient proxy to establish whether a solute will stabilize or destabilize domain phase separation. Combined with umbrella sampling simulations and coarse-grained molecular dynamics simulations, we are able to screen solutes across a wide range of chemistries and polarities. Our results indicate that, for the system under consideration, preferential partitioning and therefore effectiveness in altering membrane phase separation are strictly linked to the location of insertion in the bilayer (i.e., midplane or interface). Our approach represents a fast and simple tool for obtaining structural and thermodynamic insight into the partitioning of small molecules between lipid domains and its relation to phase separation, ultimately providing a platform for identifying the key determinants of this process.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 4 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Carleen Kluger, Lukas Braun, Steffen M. Sedlak, Diana A. Pippig, Magnus S. Bauer, Ken Miller, Lukas F. Milles, Hermann E. Gaub, Viola Vogel〈/p〉

Description: 〈p〉Publication date: Available online 1 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): P. Adhyapak, A.T. Srivatsav, M. Mishra, A. Singh, R. Narayan, S. Kapoor〈/p〉 〈h5〉Summary〈/h5〉 〈div〉〈p〉〈em〉Mycobacterium〈/em〉 species〈em〉,〈/em〉 including 〈em〉Mycobacterium tuberculosis (Mtb)〈/em〉, employs atypical long (C〈sub〉60–90〈/sub〉) and branched lipids to produce a complex cell wall and localizes these towards distinct spatial locations, inner membrane (IM) and outer membrane (OM), thus forming a robust permeability barrier. The properties and functional roles of these spatially orchestrated membrane platforms remain unknown. Herein, we report the distinctive lateral organization, fluidity and lipid domain architecture of protein-free membranes reconstituted from IM and OM lipids 〈em〉in vitro〈/em〉 from 〈em〉M〈/em〉. 〈em〉smegmatis (Msm)〈/em〉 underscored by their lipid packing and lipid dynamics. We show that 〈em〉Msm〈/em〉 OM, against common notion, is more dynamic and fluid compared with IM, and reveal the role of cell-wall associated peptidoglycans and lipoarabinomannon on the 〈em〉Msm〈/em〉 OM organization. Overall, these studies indicate that mycobacterial species may regulate their overall membrane functionality by regulating the synthesis of these complex arrays of lipids. Based on the structure-function relationship drawn here, documented alteration in the mycobacterial lipidome during cellular infection and/or drug treatment could reflect a mechanism to fine-tune 〈em〉Mtb〈/em〉 membrane properties to its advantage. These findings are expected to inspire development of lipid-centric therapeutic approaches targeted towards its membrane.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 1 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Hunter C. Davis, Sunghwi Kang, Jae-Hyun Lee, Tae-Hyun Shin, Harry Putterman, Jinwoo Cheon, Mikhail G. Shapiro〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radiofrequency alternating magnetic fields have generated intense interest due to the potential utility of this phenomenon for non-invasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from classical heat transfer theory. Here we report an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radiofrequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface, nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 1 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Edward H. Egelman〈/p〉

Description: 〈p〉Publication date: Available online 1 February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Robert S. Eisenberg〈/p〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): K. Kulenkampff, A.H. Lippert, J. McColl, M.A. Santos, A. Ponjavic, Edward Jenkins, Jane Humphrey, Alexander Winkel, Kristian Franze, S.F. Lee, S.J. Davis, D. Klenerman〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Cell-cell contacts often underpin signalling between cells. For immunology the binding of a T cell receptor (TCR) to an antigen presenting pMHC initiates downstream signalling and an immune response. While this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many often contradictory observations have been made regarding the influence of the contact on parameters such as the binding kinetics, spatial distribution and diffusion of signalling proteins within the contact. Understanding the basic physical constraints on probes inside this crowded environment will help inform studies on binding kinetics and dynamics of signalling of relevant proteins in the synapse. By tracking quantum dots of different dimensions for extended periods of time, we have shown that it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry and the impact of spatial heterogeneity of adhesion protein density in the contact. By analysing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observed probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous, due to topography- and protein density-related processes. These effects are likely to profoundly influence signalling between cells.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): A.D. Skinkle, K.R. Levental, I. Levental〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Giant Plasma Membrane Vesicles (GPMVs) are a widely used experimental platform for biochemical and biophysical analysis of isolated mammalian plasma membranes (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed; i.e. that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic Giant Unilamellar Vesicles (GUVs). We attribute this leakiness to stable, relatively large, and heterogeneous pores formed by rupture of vesicles from cells. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration important for interpreting experiments utilizing GPMVs as plasma membrane models, particularly for drug permeation and membrane asymmetry.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Abhishek A. Kognole, Alexander D. MacKerell〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg〈sup〉2+〈/sup〉, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg〈sup〉2+〈/sup〉 in folded conformations of RNA have been studied extensively, however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) simulations are used to capture the energetics and atomic-level details of Mg〈sup〉2+〈/sup〉-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg〈sup〉2+〈/sup〉, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg〈sup〉2+〈/sup〉 with two or more non-sequential phosphate groups. Notably, the present results indicate a push-pull mechanism where the Mg〈sup〉2+〈/sup〉-RNA interactions actually lead to destabilization of specific non-sequential phosphate-phosphate interactions (i.e. pushed apart) while other interactions are stabilized (i.e. pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister including formation hydrogen bonds associated with the tertiary structure. The present study establishes a better understanding of how Mg〈sup〉2+〈/sup〉 ion-interactions contribute to RNA structural properties and stability.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 17 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Jihan Kim, Yu Zheng, Amani A. Alobaidi, Hanqing Nan, Jianxiang Tian, Yang Jiaoyang, Bo Sun〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Metastasis of mesenchymal tumor cells is traditionally considered as a single cell process. Here we report an emergent collective phenomenon where the dissemination rate of mesenchymal breast cancer cells from 3D tumors depends on the tumor geometry. Combining experimental measurements and computational modeling we demonstrate that the collective dynamics is coordinated by the mechanical feedback between individual cells and their surrounding extracellular matrix (ECM). We find the tissue-like fibrous ECM supports long-range physical interactions between cells, which turn geometric cues into regulated cell dissemination dynamics. Our results suggest that migrating cells in 3D ECM represent a distinct class of active particle system where the collective dynamics is governed by remodeling of the environment rather than direct particle-particle interactions.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 16 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): D.W. Allender, Ha. Giang, M. Schick〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉We consider a model lipid plasma membrane, one that describes the outer leaf as consisting of sphingomyelin, phosphatidylcholine, and cholesterol, and the inner leaf of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and cholesterol. Their relative compositions are taken from experiment; the cholesterol freely interchanges between leaves. Fluctuations in local composition are coupled to fluctuations in the local membrane curvature, as in the Leibler-Andelman mechanism. Structure factors of components in both leaves display a peak at non-zero wavevector. This indicates that the disordered fluid membrane is characterized by structure of the corresponding wavelength. The scale is given by membrane properties: its bending modulus and its surface tension, that arises from the membrane’s connections to the cytoskeleton. From measurements on the plasma membrane, this scale is on the order of 100 nm. We find that the membrane can be divided into two different kinds of domains that differ not only in their composition, but also in their curvature. The first domain in the outer, exoplasmic, leaf is rich in cholesterol and sphingomyelin, while the inner, cytoplasmic, leaf is rich in phosphatidylserine and phosphatidylcholine. The second kind of domain is rich in phosphatidylcholine in the outer leaf, and in cholesterol and phosphatidylethanolamine in the inner leaf. The theory provides a tenable basis for the origin of structure in the plasma membrane, and an illuminating picture of the organization of lipids therein.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 16 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Sarah Jacqueline Moe, Alessandro Cembran〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin’s spectrin repeat 1, and related the changes in the protein’s structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically-induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein’s small protrusions in structurally ordered waters that are characteristic of the so-called “small-molecule regime”, which allow water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically-induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 16 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Fulin Xing, Songyue Qu, Junfang Liu, Jianyu Yang, Fen Hu, Irena Drevenšek-Olenik, Leiting Pan, Jingjun Xu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca〈sup〉2+〈/sup〉 communication between post-mitotic cells. In the present work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca〈sup〉2+〈/sup〉 signal transfer over the intercellular bridges. With use of such micropatterns considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca〈sup〉2+〈/sup〉 signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca〈sup〉2+〈/sup〉 diffusion and endogenous Ca〈sup〉2+〈/sup〉 response in the intercellular bridge-connected cell, indicating two different pathways of the Ca〈sup〉2+〈/sup〉 communication. Extracellular Ca〈sup〉2+〈/sup〉 and the paracrine pathway have practically no effect on the endogenous Ca〈sup〉2+〈/sup〉 response demonstrated by application of Ca〈sup〉2+〈/sup〉-free medium, exogenous ATP and P2Y〈sub〉13〈/sub〉 receptor antagonist. In contrast, endoplasmic reticulum Ca〈sup〉2+〈/sup〉-ATPase inhibitor thapsigargin and inositol trisphosphate (IP〈sub〉3〈/sub〉) receptor blocker 2-aminoethyl diphenylborate (2-APB) significantly inhibit the endogenous Ca〈sup〉2+〈/sup〉 increase, which signifies involvement of IP〈sub〉3〈/sub〉-sensitive calcium store release. Notably, passive Ca〈sup〉2+〈/sup〉 diffusion into the connected cell can clearly be detected when IP〈sub〉3〈/sub〉-sensitive calcium store release is abolished by 2-APB. Those observations prove that both, passive Ca〈sup〉2+〈/sup〉 diffusion and IP〈sub〉3〈/sub〉-mediated endogenous Ca〈sup〉2+〈/sup〉 response contribute to the Ca〈sup〉2+〈/sup〉 increase in intercellular bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 13 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Marco Linari, Gabriella Piazzesi, Irene Pertici, Jody A. Dantzig, Yale E. Goldman, Vincenzo Lombardi〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments during cyclical ATP-driven interactions toward the center of the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here, we review some old and new data that confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. The implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in nonmuscle intracellular cargo transport.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 11 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Christophe Jardin, Gustavo Chaves, Boris Musset〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Voltage-gated proton channels (H〈sub〉V〈/sub〉1) are essential for various physiological tasks but are strongly inhibited by Zn〈sup〉2+〈/sup〉 cations. Some determinants of Zn〈sup〉2+〈/sup〉 binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn〈sup〉2+〈/sup〉 binds to monomeric H〈sub〉V〈/sub〉1 despite evidence that H〈sub〉V〈/sub〉1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn〈sup〉2+〈/sup〉 via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 〈em〉μ〈/em〉s for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human H〈sub〉V〈/sub〉1. The dimer interface is more efficient for attracting and binding Zn〈sup〉2+〈/sup〉 via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 11 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Simli Dey, Anirban Das, Sudipta Maiti〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such “prebleaching” can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B 〉 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 9 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Shahar Goren, Yoni Koren, Xinpeng Xu, Ayelet Lesman〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The unique nonlinear mechanics of the fibrous extracellular matrix (ECM) facilitates long-range cell-cell mechanical communications that would be impossible for linear elastic substrates. Past research has described the contribution of two separated effects on the range of force transmission, including ECM elastic non-linearity and fiber alignment. However, the relation between these different effects is unclear, and how they combine to dictate force transmission range is still elusive. Here, we combine discrete fiber simulations with continuum modeling to study the decay of displacements induced by a contractile cell in fibrous networks. We demonstrate that fiber non-linearity and fiber reorientation both contribute to the strain-induced elastic anisotropy of the cell’s local environment. This elastic anisotropy is a “lumped” parameter that governs the slow decay of the displacements, and it depends on the magnitude of applied strain, either an external tension or an internal contraction as a model of the cell. Furthermore, we show that accounting for artificially-prescribed elastic anisotropy dictates the decay of displacement induced by a contracting cell. Our findings unify previous single effects into a mechanical theory that explains force transmission in fibrous networks. This work may provide insight into biological processes that involve the communication of distant cells mediated by the ECM, such as that occurring in morphogenesis, wound healing, angiogenesis, and cancer metastasis. It may also provide design parameters for biomaterials to control force transmission between cells, as a way to guide morphogenesis in tissue engineering.〈/p〉〈/div〉

Description: 〈p〉Publication date: 21 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal, Volume 118, Issue 2〈/p〉 〈p〉Author(s): Priyanka Prakash, Douglas Litwin, Hong Liang, Suparna Sarkar-Banerjee, Drew Dolino, Yong Zhou, John F. Hancock, Vasanthi Jayaraman, Alemayehu A. Gorfe〈/p〉

Description: 〈p〉Publication date: Available online 3 January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Biophysical Journal〈/p〉 〈p〉Author(s): Giulia Salzano, Martha Brennich, Giordano Mancini, Thanh Hoa Tran, Giuseppe Legname, Paola D’Angelo, Gabriele Giachin〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Prions are pathological isoforms of the cellular prion protein that is responsible for transmissible spongiform encephalopathies (TSE). Cellular prion protein interacts with copper, Cu(II), through octarepeat and nonoctarepeat (non-OR) binding sites. The molecular details of Cu(II) coordination within the non-OR region are not well characterized yet. By the means of small angle x-ray scattering and x-ray absorption spectroscopic methods, we have investigated the effect of Cu(II) on prion protein folding and its coordination geometries when bound to the non-OR region of recombinant prion proteins (recPrP) from mammalian species considered resistant or susceptible to TSE. As the prion resistant model, we used ovine recPrP (OvPrP) carrying the protective polymorphism at residues A136, R154, and R171, whereas as TSE-susceptible models, we employed OvPrP with V136, R154, and Q171 polymorphism and bank vole recPrP. Our analysis reveals that Cu(II) affects the structural plasticity of the non-OR region, leading to a more compacted conformation. We then identified two Cu(II) coordination geometries: in the type 1 coordination observed in OvPrP at residues A136, R154, and R171, the metal is coordinated by four residues; conversely, the type 2 coordination is present in OvPrP with V136, R154, and Q171 and bank vole recPrP, where Cu(II) is coordinated by three residues and by one water molecule, making the non-OR region more exposed to the solvent. These changes in copper coordination affect the recPrP amyloid aggregation. This study may provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.〈/p〉〈/div〉