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  • Thermal and Chemical Unfolding of a monoclonal IgG1 antibody: Application of the Multi-State Zimm-Bragg Theory
    Biophys. J. (IF 3.665) Pub Date : 2020-01-16
    P. Garidel; A. Eiperle; M. Blech; J. Seelig

    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 ΔHcal ∼ 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 ∼6x10−5 and a Gibbs free energy of unfolding of gnu ∼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 ΔHcal and lowers the midpoint temperature Tm. 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.

    更新日期:2020-01-17
  • Mechanical Unfolding of Spectrin Repeats Induces Water-Molecule Ordering
    Biophys. J. (IF 3.665) Pub Date : 2020-01-16
    Sarah Jacqueline Moe; Alessandro Cembran

    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.

    更新日期:2020-01-17
  • Model Plasma Membrane exhibits a Microemulsion in both Leaves providing a Foundation for “Rafts”
    Biophys. J. (IF 3.665) Pub Date : 2020-01-16
    D.W. Allender; Ha. Giang; M. Schick

    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.

    更新日期:2020-01-17
  • Intercellular bridge mediates Ca2+ signals between micropatterned cells via IP3 and Ca2+ diffusion
    Biophys. J. (IF 3.665) Pub Date : 2020-01-16
    Fulin Xing; Songyue Qu; Junfang Liu; Jianyu Yang; Fen Hu; Irena Drevenšek-Olenik; Leiting Pan; Jingjun Xu

    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 Ca2+ 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 Ca2+ 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 Ca2+ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca2+ diffusion and endogenous Ca2+ response in the intercellular bridge-connected cell, indicating two different pathways of the Ca2+ communication. Extracellular Ca2+ and the paracrine pathway have practically no effect on the endogenous Ca2+ response demonstrated by application of Ca2+-free medium, exogenous ATP and P2Y13 receptor antagonist. In contrast, endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and inositol trisphosphate (IP3) receptor blocker 2-aminoethyl diphenylborate (2-APB) significantly inhibit the endogenous Ca2+ increase, which signifies involvement of IP3-sensitive calcium store release. Notably, passive Ca2+ diffusion into the connected cell can clearly be detected when IP3-sensitive calcium store release is abolished by 2-APB. Those observations prove that both, passive Ca2+ diffusion and IP3-mediated endogenous Ca2+ response contribute to the Ca2+ increase in intercellular bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.

    更新日期:2020-01-17
  • Nucleosomal DNA dynamics mediate Oct4 pioneer factor binding
    Biophys. J. (IF 3.665) Pub Date : 2020-01-16
    Jan Huertas; Caitlin M. MacCarthy; Hans R. Schöler; Vlad Cojocaru

    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.

    更新日期:2020-01-17
  • Modulating the Stiffness of the Myosin-VI Single α-Helical Domain
    Biophys. J. (IF 3.665) Pub Date : 2020-01-15
    C. Ashley Barnes; Yang Shen; Jinfa Ying; Ad Bax

    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 ca 20% of the time, two consecutive Gly substitutions break the helix for ca 65% of the time. NMR relaxation measurements indicate that the exchange rate between intact and broken helix is fast (>300,000 s-1) 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.

    更新日期:2020-01-15
  • Nanosecond Timescale Dynamics and Conformational Heterogeneity in Human Glucokinase Regulation and Disease
    Biophys. J. (IF 3.665) Pub Date : 2020-01-14
    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

    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 19F 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.

    更新日期:2020-01-15
  • Straightening out the elasticity of myosin cross-bridges
    Biophys. J. (IF 3.665) Pub Date : 2020-01-13
    M. Linari; G. Piazzesi; I. Pertici; J.A. Dantzig; Y.E. Goldman; V. Lombardi

    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 which confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. Implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in non-muscle intracellular cargo transport.

    更新日期:2020-01-14
  • Correction of systematic bias in single molecule photo-bleaching measurements
    Biophys. J. (IF 3.665) Pub Date : 2020-01-11
    Simli Dey; Anirban Das; Sudipta Maiti

    Single molecule photobleaching (smPB) is a powerful technique to measure the number of fluorescent units in sub-resolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photo-bleaching can occur before the sample is appropriately placed and focused. Such ‘pre-bleaching’ can introduce a strong systematic bias towards smaller oligomers. Quantitative correction of pre-bleaching 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 pre-bleaching 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 (IAPP). 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.

    更新日期:2020-01-11
  • Assessing structural determinants of Zn2+ binding to human HV1 via multiple MD simulations
    Biophys. J. (IF 3.665) Pub Date : 2020-01-11
    Christophe Jardin; Gustavo Chaves; Boris Musset

    Voltage-gated proton channels (HV1) are essential for various physiological tasks but are strongly inhibited by Zn2+ cations. Some determinants of Zn2+ binding have been elucidated, experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn2+ binds to monomeric HV1 despite evidence that HV1 expresses as a dimer, that the dimer has a higher affinity for zinc than the monomer, and experimental data that suggests 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 series of molecular dynamics simulations to address different structures of the human proton channel (hHV1), the monomer and two plausible dimer conformations to compare their respective potential to interact with and bind Zn2+ 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 μs 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 hHV1. The dimer interface is more efficient to attract and bind Zn2+ 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 towards the binding sites.

    更新日期:2020-01-11
  • Exploring Conformational Change of Adenylate Kinase by Replica Exchange Molecular Dynamic Simulation
    Biophys. J. (IF 3.665) Pub Date : 2020-01-09
    Jinan Wang; Cheng Peng; Yuqu Yu; Zhaoqiang Chen; Zhijian Xu; Tingting Cai; Qiang Shao; Jiye Shi; Weiliang Zhu

    Replica exchange molecular dynamics (REMD) simulation is a popular enhanced sampling method, which is widely used for exploring atomic mechanism of protein conformational change. However, the requirement of huge computational resources for REMD, especially with explicit solvent model, largely limits its application. In this study the availability and efficiency of a variant of velocity-scaling replica exchange molecular dynamics (vsREMD) was assessed with adenylate kinase (AdK) as an example. While vsREMD achieved consistent results with that from conventional REMD and experimental studies, the number of replicas required for vsREMD (30) was much less than that for conventional REMD (80) to achieve similar acceptance rate (∼0.2), demonstrating high efficiency of vsREMD to characterize protein conformational change and associated free energy profile. Thus, vsREMD is a highly efficient approach for studying large-scale conformational change of protein systems.

    更新日期:2020-01-09
  • Elastic Anisotropy Governs the Range of Cell-induced Displacements
    Biophys. J. (IF 3.665) Pub Date : 2020-01-09
    Shahar Goren; Yoni Koren; Xinpeng Xu; Ayelet Lesman

    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.

    更新日期:2020-01-09
  • Structural consequences of multisite phosphorylation in the BAK1 kinase domain
    Biophys. J. (IF 3.665) Pub Date : 2020-01-03
    Alexander S. Moffett; Diwakar Shukla

    Multisite phosphorylation is an important mechanism of post-translational control of protein kinases. The effects of combinations of possible phosphorylation states on protein kinase activity are difficult to study experimentally due to challenges in isolating a particular phosphorylation state, while surprising little effort on this topic has been expended in computational studies. In order to understand the effects of multisite phosphorylation on the plant protein kinase BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) conformational ensemble, we performed Gaussian accelerated molecular dynamics simulations on eight BAK1 mod-forms involving phosphorylation of the four activation loop threonine residues and binding of ATP-Mg2+. We find that unphosphorylated BAK1 transitions into an inactive conformation with a “cracked” activation loop and with the αC helix swung away from the active site. T450 phosphorylation can prevent the activation loop from cracking and keep the αC helix in an active-like conformation, while phosphorylation of T455 only slightly stabilizes the activation loop. There is a general trend of reduced flexibility in inter-lobe motion with increased phosphorylation. Interestingly, the αC helix is destabilized when the activation loop is fully phosphorylated but is again stabilized with ATP-Mg2+ bound. Our results provide insight into the mechanism of phosphorylation-controlled BAK1 activation while at the same time representing, to our knowledge, the first comprehensive, comparative study of the effects of combinatorial phosphorylation states on protein kinase conformational dynamics.

    更新日期:2020-01-04
  • Quantitative Analysis of Spatial Distributions of all tRNA Genes in Budding Yeast
    Biophys. J. (IF 3.665) Pub Date : 2020-01-03
    Naoko Tokuda

    In the budding yeast nucleus, transfer RNA (tRNA) genes are considered to localize in the vicinity of the nucleolus; however, the use of Hi-C and the fluorescent repressor-operator system techniques has clearly indicated that the tRNA genes are distributed not only around the nucleolus but also at other nuclear locations. However, there are some discrepancies between Hi-C data analysis and the results indicated from fluorescence microscopy data. To fill these gaps, we systematically clarified the spatial arrangements of all tRNA genes in the budding yeast nucleus using the genome simulation model developed by us. The simulation results revealed that out of 275 tRNA genes, 58% were found to be spatially distributed around the centromeres, 16% were distributed around the ribosomal DNA (rDNA) regions, and the remaining 26% were distributed between the centromeres and rDNA regions. Furthermore, 1% of all tRNA genes were found to be spatially distributed around the nuclear envelope, 30% were distributed around the center of the nucleus, and the remaining 69% were distributed between the nuclear envelope and the center of the nucleus. The percentage distributions were highly similar to those of the 176 tRNA genes encoding tRNAs having an anticodon for the optimal codons. The simulation results also revealed that the spatial arrangements of tRNA genes were affected by linear genomic distance from the tethering elements, such as the centromeres or telomeres; however, the distance was only one of the factors to determine spatial distribution. This study, also investigates whether tRNA gene transcriptional levels depend on the arrangements in the budding yeast nucleus by integrating the genome simulation model with tRNA-seq data. The results suggest that the transcriptional levels did not depend on the arrangements in the nucleus. By using the genome simulation model, we showed the possibility to quantitatively analyze genome structures.

    更新日期:2020-01-04
  • Study of the expression transition of cardiac myosin using polarization-dependent SHG microscopy
    Biophys. J. (IF 3.665) Pub Date : 2020-01-03
    Cai Yuan; Xiaolei Zhao; Zhonghai Wang; Thomas K. Borg; Tong Ye; Zain I. Khalpey; Raymond B. Runyan; Yonghong Shao; Bruce Z. Gao

    Detection of the transition between the two myosin isoforms alpha- and beta-myosin in living cardiomyocytes is essential for understanding cardiac physiology and pathology. In this study, the differences in symmetry of polarization spectra obtained from alpha- and beta-myosin in various mammalian ventricles and propylthiouracil-treated rats are explored through polarization-dependent second harmonic generation microscopy. Here, we report for the first time that alpha- and beta-myosin, as protein crystals, possess different symmetries: The former has C6 symmetry, and the latter, C3v. A single sarcomere line scan further demonstrated that the differences in polarization-spectrum symmetry between alpha- and beta-myosin came from their head regions: The head and neck domains of alpha- and beta-myosin account for the differences in symmetry. In addition, the dynamic transition of the polarization spectrum from C6 to C3v line profile was observed in a cell culture in which norepinephrine induced an alpha- to beta-myosin transition.

    更新日期:2020-01-04
  • Primary and secondary binding ofPrimary and secondary peptide binding exenatide to liposomes
    Biophys. J. (IF 3.665) Pub Date : 2020-01-03
    Anja Stulz; Michaela Breitsamer; Gerhard Winter; Heiko H. Heerklotz

    The interactions of exenatide, a tryptophan-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis (MST) measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel (VPG) formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide–lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phos phatidyl glycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (Kd = 46 μM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per 5 PG molecules with a Kd ≈ 0.2 μM for PC/PG 1:1 and 0.6 μM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light scattering measurements. ITC quantifies primary and secondary binding separately, whereas MST and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here.

    更新日期:2020-01-04
  • Deciphering copper coordination in the mammalian prion protein amyloidogenic domain
    Biophys. J. (IF 3.665) Pub Date : 2020-01-03
    Giulia Salzano; Martha Brennich; Giordano Mancini; Thanh Hoa Tran; Giuseppe Legname; Paola D’Angelo; Gabriele Giachin

    Prions are pathological isoforms of the cellular prion protein (PrPC) responsible for transmissible spongiform encephalopathies (TSE). PrPC interacts with copper, Cu(II), through octarepeat and non-octarepeat (non-OR) binding sites. The molecular details of Cu(II) coordination within the non-OR region are not well characterized yet. By means of small angle X-ray scattering (SAXS) and X-ray absorption spectroscopic methods (XAS) 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 prion resistant model, we used ovine recPrP (OvPrP) carrying the protective polymorphism at residues A136, R154 and R171 (ARR); while as TSE-susceptible models we employed OvPrP with V136, R154 and Q171 (VRQ) polymorphism and Bank vole recPrP (BvPrP). 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 ARR the metal is coordinated by four residues; conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, 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.

    更新日期:2020-01-04
  • Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP
    Biophys. J. (IF 3.665) Pub Date : 2020-01-03
    Zachary Ridgway; Kyung-Hoon Lee; Alexander Zhyvoloup; Amy Wong; Charles Eldrid; Eleni Hannaberry; Konstantinos Thalassinos; Andisheh Abedini; Daniel P. Raleigh

    The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic β-cell dysfunction and death. Not all species form islet amyloid and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a non-conservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same time scale as human amylin in vitro and exhibits similar toxicity towards cultured β-cells. The K1I replacement in human amylin slightly reduces toxicity, while the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveal that the baboon amylin, like human amylin, forms low order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Pre amyloid oligomers formed by baboon amylin, but not baboon amylin fibers are toxic to cultured β-cells. The toxicity of baboon oligomers and lack of significant detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that pre-amyloid oligomers are the most toxic species produced during IAPP amyloid formation.

    更新日期:2020-01-04
  • Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization
    Biophys. J. (IF 3.665) Pub Date : 2019-12-31
    Anisha Shakya; Seonyoung Park; Neha Rana; John T. King

    Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membrane-less 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 absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in presence of DNA and nucleosomes in vitro, while 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 HP1α), 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. While LLPS of HP1α 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.

    更新日期:2020-01-01
  • Is the E. coli homolog of the formate/nitrite transporter family an anion channel? A computational study
    Biophys. J. (IF 3.665) Pub Date : 2019-12-31
    Mishtu Mukherjee; Ankita Gupta; Ramasubbu Sankararamakrishnan

    Formate/Nitrite Transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lowers eukaryotes. They play significant role in bacterial growth and act against the defense mechanism of infected host. Since FNTs don’t occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups and two of which belong to the uncharacterized YfdC-α and YfdC-β groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered formate channel from Vibrio Cholerae (VcFocA), hydrosulphide channel from Clostridium difficile (CdHSC) and the uncharacterized channel from Escherichia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics (MD) and umbrella sampling studies, we determined temporal channel radius profiles, permeation events and potential of mean force (PMF) profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the VcFocA and CdHSC, MD studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-α, were found to be responsible for high energy barriers for the anions to enter EcYfdC. PMF profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies strongly suggest that EcYfdC is not an anion channel. Absence/presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. Further experimental studies are needed to get a definite answer to the question of the substrate selectivity of EcYfdC. This provides an example of membrane proteins from the same family transporting substrates of different chemical nature.

    更新日期:2020-01-01
  • Membrane and actin tethering transitions help IQGAP1 coordinate GTPase and lipid messenger signaling
    Biophys. J. (IF 3.665) Pub Date : 2019-12-31
    Nicholaus Trenton; R. Tyler McLaughlin; Satya K. Bellamkonda; David S. Tsao; Alexandra Rodzinski; Emily M. Mace; Jordan S. Orange; Volker Schweikhard; Michael R. Diehl

    The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multi-domain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live-cell imaging to probe how the multi-domain scaffold IQGAP1 coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal-membrane tethered states. Fast scaffold binding / disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. While this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain (CHD) of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) colocalization analyses or traditional pathway perturbations methods, and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase PI3K in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.

    更新日期:2020-01-01
  • Micro-scale fluid behavior during cryo-EM sample blotting
    Biophys. J. (IF 3.665) Pub Date : 2019-12-25
    Maxim Armstrong; Bong-Gyoon Han; Salvador Gomez; John Turner; Daniel A. Fletcher; Robert M. Glaeser

    Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy (cryo-EM) for over three decades. This technique removes excess solution from a TEM grid by pressing absorbent filter paper against the specimen prior to vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference-contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topolgraphical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses.

    更新日期:2019-12-26
  • Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms
    Biophys. J. (IF 3.665) Pub Date : 2019-12-25
    Raya Sorkin; Margherita Marchetti; Emma Logtenberg; Melissa Piontek; Emma Kerklingh; Guy Brand; Rashmi Voleti; Josep Rizo; Wouter H. Roos; Alexander J. Groffen; Gijs J.L. Wuite

    Synaptotagmin-1 is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically-trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared to that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane bending modulus, as measured by an AFM-based method. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

    更新日期:2019-12-26
  • Magnetic Susceptibility Difference-induced Nucleus Positioning in Gradient Ultra-high Magnetic Field
    Biophys. J. (IF 3.665) Pub Date : 2019-12-25
    Qingping Tao; Lei Zhang; Xuyao Han; Hanxiao Chen; Xinmiao Ji; Xin Zhang

    Despite the importance of magnetic properties of biological samples for biomagnetism and related fields, the exact magnetic susceptibilities of most biological samples in their physiological conditions are still unknown. Here we used SQUID (Superconducting Quantum Interferometer Device) to detect the magnetic properties of non-fixed non-dehydrated live cell and cellular fractions, at a physiological temperature of 37 °C (310 K). It is obvious that there are paramagnetic components within human nasopharyngeal carcinoma CNE-2Z cells. More importantly, the magnetic properties of cytoplasm and nucleus are different. Although within a single cell, the magnetic susceptibility difference between cellular fractions (nucleus and cytoplasm) could only cause ∼41-130 pN forces to nucleus by gradient ultra-high magnetic fields of 13.1-23.5 T, 92-160 T/m, these forces are enough to cause a relative position shift of the nucleus within the cell. This not only demonstrates the importance of magnetic susceptibility in the biological effects of magnetic field, but also illustrates the potential application of high magnetic fields in biomedicine.

    更新日期:2019-12-26
  • Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation
    Biophys. J. (IF 3.665) Pub Date : 2019-12-25
    Ivo Kabelka; Michael Pachler; Sylvain Prévost; Ilse Letofsky-Papst; Karl Lohner; Georg Pabst; Robert Vácha

    We studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding paper [Pachler et al., Biophys. J. 2019, 117, 1858–1869xxx], we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in membranes. Here, we focus on the structures of the peptide/lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles, with a collapsed interbilayer spacing resulting from peptide induced adhesion. Interestingly, the adhesion does not lead to a peptide induced lipid separation of charged and charge neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle X-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.

    更新日期:2019-12-26
  • Measurement of skeletal muscle fiber contractility with high-speed traction microscopy
    Biophys. J. (IF 3.665) Pub Date : 2019-12-24
    Martin Rausch; David Böhringer; Martin Steinmann; Dirk W. Schubert; Stefan Schrüfer; Christoph Mark; Ben Fabry

    We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young’s modulus of the matrix, without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/ml Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 μN ± 0.15 μN and tetanic forces (100 Hz stimulation frequency) of 2.38 μN ± 0.71 μN, corresponding to a tension of 0.44 kPa ± 0.25 kPa and 2.53 kPa ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium-stabilizer Tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

    更新日期:2019-12-25
  • Molecular insights from conformational ensembles via machine learning
    Biophys. J. (IF 3.665) Pub Date : 2019-12-21
    Oliver Fleetwood; Marina A. Kasimova; Annie M. Westerlund; Lucie Delemotte

    Biomolecular simulations are intrinsically high dimensional and generate noisy datasets of ever increasing size. Extracting important features in the data is crucial for understanding the biophysical properties of molecular processes, but remains a big challenge. Machine learning (ML) provides powerful dimensionality reduction tools. However, such methods are often criticized to resemble black boxes with limited human-interpretable insight. We use methods from supervised and unsupervised ML to efficiently create interpretable maps of important features from molecular simulations. We benchmark the performance of several methods including neural networks, random forests and principal component analysis, using a toy model with properties reminiscent of macromolecular behavior. We then analyze three diverse biological processes: conformational changes within the soluble protein calmodulin, ligand binding to a G protein-coupled receptor and activation of an ion channel voltage-sensor domain, unravelling features critical for signal transduction, ligand binding and voltage sensing. This work demonstrates the usefulness of ML in understanding biomolecular states and demystifying complex simulations.

    更新日期:2019-12-21
  • Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties
    Biophys. J. (IF 3.665) Pub Date : 2019-12-21
    Ankush Garg; Jagadish Prasad Hazra; Malay Kumar Sannigrahi; Sabyasachi Rakshit; Sharmistha Sinha

    Mutations in p53 protein, especially in the DNA binding domain is one of the major hallmarks of cancer. The R273 position is a DNA contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant-variants of R273 in p53 have different survival rates indicating that the DNA contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the properties of three major oncogenic variants of the wild type p53, [R273H]p53, [R273C]p53, and [R273L]p53. Using a series of biophysical, biochemical and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, also show distinct structural stability, aggregation and toxicity profiles. The WTp53 and the [R273H]p53 show the least destabilization and aggregation propensity. [R273C]p53 aggregation is disulphide mediated leading to cross-beta, ThT positive aggregates whereas hydrophobic interactions dominate self-assembly in [R273L]p53 leading to a mixture of amyloid and amorphous aggregates. MD simulations indicate different contact maps and secondary structures for the different variants along the course of the simulations. Our study indicates that each of the R273 variants has its own distinct property of stability and self-assembly, the molecular basis of which, may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue-specific or process specific protein aggregation as a target.

    更新日期:2019-12-21
  • A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    A. Hornung; T. Sbarrato; N. Garcia-Seyda; L. Aoun; X. Luo; M. Biarnes-Pelicot; O. Theodoly; M.P. Valignat

    Recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with mechanisms only partially deciphered. Here, we studied the guidance by flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins LFA-1 (αLβ2) and VLA-4 (α4β1). We reveal that cells segregate in two populations of opposite orientation for combined adhesion, and show that decisions of orientation rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-mediated downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrins affinity, combined with an inhibiting crosstalk of LFA-1 towards VLA-4. At the cellular level, direction is determined by the passive, flow-mediated orientation of the non-adherent cell parts, the rear uropod for upstream migration and the front lamellipod for downstream migration. This chain of logical events provides a comprehensive mechanism of guiding, from stimuli to cell orientation.

    更新日期:2019-12-19
  • Hypocalcemia-Induced Slowing of Human Sinus Node Pacemaking
    Biophys. J. (IF 3.665) Pub Date : 2019-07-30
    Axel Loewe; Yannick Lutz; Deborah Nairn; Alan Fabbri; Norbert Nagy; Noemi Toth; Xiaoling Ye; Doris H. Fuertinger; Simonetta Genovesi; Peter Kotanko; Jochen G. Raimann; Stefano Severi

    Each heartbeat is initiated by cyclic spontaneous depolarization of cardiomyocytes in the sinus node forming the primary natural pacemaker. In patients with end-stage renal disease undergoing hemodialysis, it was recently shown that the heart rate drops to very low values before they suffer from sudden cardiac death with an unexplained high incidence. We hypothesize that the electrolyte changes commonly occurring in these patients affect sinus node beating rate and could be responsible for severe bradycardia. To test this hypothesis, we extended the Fabbri et al. computational model of human sinus node cells to account for the dynamic intracellular balance of ion concentrations. Using this model, we systematically tested the effect of altered extracellular potassium, calcium, and sodium concentrations. Although sodium changes had negligible (0.15 bpm/mM) and potassium changes mild effects (8 bpm/mM), calcium changes markedly affected the beating rate (46 bpm/mM ionized calcium without autonomic control). This pronounced bradycardic effect of hypocalcemia was mediated primarily by ICaL attenuation due to reduced driving force, particularly during late depolarization. This, in turn, caused secondary reduction of calcium concentration in the intracellular compartments and subsequent attenuation of inward INaCa and reduction of intracellular sodium. Our in silico findings are complemented and substantiated by an empirical database study comprising 22,501 pairs of blood samples and in vivo heart rate measurements in hemodialysis patients and healthy individuals. A reduction of extracellular calcium was correlated with a decrease of heartrate by 9.9 bpm/mM total serum calcium (p < 0.001) with intact autonomic control in the cross-sectional population. In conclusion, we present mechanistic in silico and empirical in vivo data supporting the so far neglected but experimentally testable and potentially important mechanism of hypocalcemia-induced bradycardia and asystole, potentially responsible for the highly increased and so far unexplained risk of sudden cardiac death in the hemodialysis patient population.

    更新日期:2019-12-18
  • Characterizing Conduction Channels in Postinfarction Patients Using a Personalized Virtual Heart
    Biophys. J. (IF 3.665) Pub Date : 2019-07-22
    Dongdong Deng; Adityo Prakosa; Julie Shade; Plamen Nikolov; Natalia A. Trayanova

    Patients with myocardial infarction have an abundance of conduction channels (CC); however, only a small subset of these CCs sustain ventricular tachycardia (VT). Identifying these critical CCs (CCCs) in the clinic so that they can be targeted by ablation remains a significant challenge. The objective of this study is to use a personalized virtual-heart approach to conduct a three-dimensional (3D) assessment of CCCs sustaining VTs of different morphologies in these patients, to investigate their 3D structural features, and to determine the optimal ablation strategy for each VT. To achieve these goals, ventricular models were constructed from contrast enhanced magnetic resonance imagings of six postinfarction patients. Rapid pacing induced VTs in each model. CCCs that sustained different VT morphologies were identified. CCCs’ 3D structure and type and the resulting rotational electrical activity were examined. Ablation was performed at the optimal part of each CCC, aiming to terminate each VT with a minimal lesion size. Predicted ablation locations were compared to clinical. Analyzing the simulation results, we found that the observed VTs in each patient model were sustained by a limited number (2.7 ± 1.2) of CCCs. Further, we identified three types of CCCs sustaining VTs: I-type and T-type channels, with all channel branches bounded by scar, and functional reentry channels, which were fully or partially bounded by conduction block surfaces. The different types of CCCs accounted for 43.8, 18.8, and 37.4% of all CCCs, respectively. The mean narrowest width of CCCs or a branch of CCC was 9.7 ± 3.6 mm. Ablation of the narrowest part of each CCC was sufficient to terminate VT. Our results demonstrate that a personalized virtual-heart approach can determine the possible VT morphologies in each patient and identify the CCCs that sustain reentry. The approach can aid clinicians in identifying accurately the optimal VT ablation targets in postinfarction patients.

    更新日期:2019-12-18
  • Cardiac Metabolic Limitations Contribute to Diminished Performance of the Heart in Aging
    Biophys. J. (IF 3.665) Pub Date : 2019-07-02
    Xin Gao; Djordje G. Jakovljevic; Daniel A. Beard

    Changes in the myocardial energetics associated with aging—reductions in creatine phosphate/ATP ratio, total creatine, and ATP—mirror changes observed in failing hearts compared to healthy controls. Similarly, both aging and heart failure are associated with significant reductions in cardiac performance and maximal left ventricular cardiac power output compared with young healthy individuals. Based on these observations, we hypothesize that reductions in the concentrations of cytoplasmic adenine nucleotide, creatine, and phosphate pools that occur with aging impair the myocardial capacity to synthesize ATP at physiological free energy levels and that the resulting changes to myocardial energetic status impair the mechanical pumping ability of the heart. The purpose of this study is to test these hypotheses using an age-structured population model for myocardial metabolism in the adult female population and to determine the potential impact of reductions in key myocardial metabolite pools in causing metabolic/energetic and cardiac mechanical dysfunction associated with aging. To test these hypotheses, we developed a population model for myocardial energetics to predict myocardial ATP, ADP, creatine phosphate, creatine, and inorganic phosphate concentrations as functions of cardiac work and age in the adult female population. Model predictions support our hypotheses and are consistent with previous experimental observations. The major findings provide a novel, to our knowledge, theoretical and computational framework for further probing complex relationships between the energetics and performance of the heart with aging.

    更新日期:2019-12-18
  • Balance of Active, Passive, and Anatomical Cardiac Properties in Doxorubicin-Induced Heart Failure
    Biophys. J. (IF 3.665) Pub Date : 2019-07-29
    Alexandre Lewalle; Sander Land; Jort J. Merken; Anne Raafs; Pilar Sepúlveda; Stéphane Heymans; Jos Kleinjans; Steven A. Niederer

    Late-onset heart failure (HF) is a known side effect of doxorubicin chemotherapy. Typically, patients are diagnosed when already at an irreversible stage of HF, which allows few or no treatment options. Identifying the causes of compromised cardiac function in this patient group may improve early patient diagnosis and support treatment selection. To link doxorubicin-induced changes in cardiac cellular and tissue mechanical properties to overall cardiac function, we apply a multiscale biophysical biomechanics model of the heart to measure the plausibility of changes in model parameters representing the passive, active, or anatomical properties of the left ventricle for reproducing measured patient phenotypes. We create representative models of healthy controls (N = 10) and patients with HF induced by (N = 22) or unrelated to (N = 25) doxorubicin therapy. The model predicts that HF in the absence of doxorubicin is characterized by a 2- to 3-fold stiffness increase, decreased tension (0–20%), and ventricular dilation (of order 10–30%). HF due to doxorubicin was similar but showed stronger bias toward reduced active contraction (10–30%) and less dilation (0–20%). We find that changes in active, passive, and anatomical properties all play a role in doxorubicin-induced cardiotoxicity phenotypes. Differences in parameter changes between patient groups are consistent with doxorubicin cardiotoxicity having a greater dependence on reduced cellular contraction and less anatomical remodeling than HF not caused by doxorubicin.

    更新日期:2019-12-18
  • Surgical Aortic Valve Replacement: Are We Able to Improve Hemodynamic Outcome?
    Biophys. J. (IF 3.665) Pub Date : 2019-07-22
    Pavlo Yevtushenko; Florian Hellmeier; Jan Bruening; Sarah Nordmeyer; Volkmar Falk; Christoph Knosalla; Marcus Kelm; Titus Kuehne; Leonid Goubergrits

    Aortic valve replacement (AVR) does not usually restore physiological flow profiles. Complex flow profiles are associated with aorta dilatation, ventricle remodeling, aneurysms, and development of atherosclerosis. All these affect long-term morbidity and often require reoperations. In this pilot study, we aim to investigate an ability to optimize the real surgical AVR procedure toward flow profile associated with healthy persons. Four cases of surgical AVR (two with biological and two with mechanical valve prosthesis) with available post-treatment cardiac magnetic resonance imaging (MRI), including four-dimensional flow MRI and showing abnormal complex post-treatment hemodynamics, were investigated. All cases feature complex hemodynamic outcomes associated with valve-jet eccentricity and strong secondary flow characterized by helical flow and recirculation regions. A commercial computational fluid dynamics solver was used to simulate peak systolic hemodynamics of the real post-treatment outcome using patient-specific MRI measured boundary conditions. Then, an attempt to optimize hemodynamic outcome by modifying valve size and orientation as well as ascending aorta size reduction was made. Pressure drop, wall shear stress, secondary flow degree, helicity, maximal velocity, and turbulent kinetic energy were evaluated to characterize the AVR hemodynamic outcome. The proposed optimization strategy was successful in three of four cases investigated. Although no single parameter was identified as the sole predictor for a successful flow optimization, downsizing of the ascending aorta in combination with the valve orientation was the most effective optimization approach. Simulations promise to become an effective tool to predict hemodynamic outcome. The translation of these tools requires, however, studies with a larger cohort of patients followed by a prospective clinical validation study.

    更新日期:2019-12-18
  • Rapid Characterization of hERG Channel Kinetics I: Using an Automated High-Throughput System
    Biophys. J. (IF 3.665) Pub Date : 2019-07-25
    Chon Lok Lei; Michael Clerx; David J. Gavaghan; Liudmila Polonchuk; Gary R. Mirams; Ken Wang

    Predicting how pharmaceuticals may affect heart rhythm is a crucial step in drug development and requires a deep understanding of a compound’s action on ion channels. In vitro hERG channel current recordings are an important step in evaluating the proarrhythmic potential of small molecules and are now routinely performed using automated high-throughput patch-clamp platforms. These machines can execute traditional voltage-clamp protocols aimed at specific gating processes, but the array of protocols needed to fully characterize a current is typically too long to be applied in a single cell. Shorter high-information protocols have recently been introduced that have this capability, but they are not typically compatible with high-throughput platforms. We present a new 15 second protocol to characterize hERG (Kv11.1) kinetics, suitable for both manual and high-throughput systems. We demonstrate its use on the Nanion SyncroPatch 384PE, a 384-well automated patch-clamp platform, by applying it to Chinese hamster ovary cells stably expressing hERG1a. From these recordings, we construct 124 cell-specific variants/parameterizations of a hERG model at 25°C. A further eight independent protocols are run in each cell and are used to validate the model predictions. We then combine the experimental recordings using a hierarchical Bayesian model, which we use to quantify the uncertainty in the model parameters, and their variability from cell-to-cell; we use this model to suggest reasons for the variability. This study demonstrates a robust method to measure and quantify uncertainty and shows that it is possible and practical to use high-throughput systems to capture full hERG channel kinetics quantitatively and rapidly.

    更新日期:2019-12-18
  • Spontaneous curvature, differential stress, and bending modulus of asymmetric lipid membranes
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Amirali Hossein; Markus Deserno

    Lipid bilayers can exhibit asymmetric states in which the physical characteristics of one leaflet differ from those of the other. This most visibly manifests in a different lipid composition, but it can also involve opposing lateral stresses in each leaflet that combine to an overall vanishing membrane tension. Here we use theoretical modeling and coarse-grained simulation to explore the interplay between a compositional asymmetry and a nonvanishing differential stress. Minimizing the total elastic energy leads to a preferred spontaneous curvature that balances torques due to both bending moments and differential stress, with sometimes unexpected consequences. For instance, asymmetric flat bilayers, whose specific areas in each leaflet are matched to those of corresponding tensionless symmetric flat membranes, still exhibit a residual differential stress, because the conditions of vanishing area strain and vanishing bending moment differ. We also measure the curvature rigidity of asymmetric bilayers and find that a sufficiently strong differential stress, but not compositional asymmetry alone, can increase the bending modulus. The likely cause is a stiffening of the compressed leaflet, which appears to be related to its gel transition, but not identical with it. We finally show that the impact of cholesterol on differential stress depends on the relative strength of elastic and thermodynamic driving forces: if cholesterol solvates equally well in both leaflets, it will redistribute to cancel both leaflet tensions almost completely; but if its partitioning free energy prefers one leaflet over the other, the resulting distribution bias may even create differential stress. Since cells keep most of their lipid bilayers in an asymmetric nonequilibrium steady state, our findings suggests that biomembranes are elastically more complex than previously thought: besides a spontaneous curvature they might also exhibit significant differential stress, which could strongly affect their curvature energetics.

    更新日期:2019-12-18
  • Rapid Characterization of hERG Channel Kinetics II: Temperature Dependence
    Biophys. J. (IF 3.665) Pub Date : 2019-07-25
    Chon Lok Lei; Michael Clerx; Kylie A. Beattie; Dario Melgari; Jules C. Hancox; David J. Gavaghan; Liudmila Polonchuk; Ken Wang; Gary R. Mirams

    Ion channel behavior can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing, inactivating, and recovery) are commonly represented with Q10 coefficients or an Eyring relationship. In this article, we assess the validity of these representations by characterizing channel kinetics at multiple temperatures. We focus on the human Ether-à-go-go-Related Gene (hERG) channel, which is important in drug safety assessment and commonly screened at room temperature so that results require extrapolation to physiological temperature. In Part I of this study, we established a reliable method for high-throughput characterization of hERG1a (Kv11.1) kinetics, using a 15-second information-rich optimized protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using Chinese hamster ovary cells overexpressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch-clamp platform, with temperature control. We characterize the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37°C and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients; it broadly follows a generalized, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol dependent. Our results show that a direct fit using our 15-s protocol best represents hERG kinetics at any given temperature and suggests that using the Generalized Eyring theory is preferable if no experimental data are available to derive model parameters at a given temperature.

    更新日期:2019-12-18
  • The chiral looping of the embryonic heart is formed by the combination of three axial asymmetries
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Hisao Honda; Takaya Abe; Toshihiko Fujimori

    In mammals and birds, embryonic development of the heart involves conversion of a straight tubular structure into a three-dimensional helical loop, which is a chiral structure. We investigated theoretically the mechanism of helical loop formation of the mouse embryonic heart, especially determination of left/right handedness of the helical loop. In geometrical terms, chirality is the result of the combination of three axial asymmetries in three-dimensional space. We hypothesized the following correspondences between axial asymmetries and morphogenesis (bending and displacement): The dorsal–ventral asymmetry by ventral bending of a straight tube of the initial heart; The left–right and anterior–posterior asymmetries, the left–right asymmetry by rightward displacement of the heart tube, which is confined to the anterior region of the tube. Morphogenesis of chiral looping of the embryonic heart is a large-scaled event of the multi-cellular system in which substantial physical force operates dynamically. Using computer simulations with a cell-based physico-mechanical model and experiments with mouse embryos, we confirmed the hypothesis. We conclude that rightward displacement of the tube determines the left-handed screw of the loop. The process of helix loop formation consists of three steps: (1) the left–right biasing system involving Nodal-related signals that leads to left–right asymmetry in the embryonic body; (2) the rightward displacement of the tube; and (3) finally the left-handed helical looping. Step 1 is already established. Step 3 is elucidated by our present study, which highlights the need for step 2 to be clarified, namely, how does the left–right asymmetry in the embryonic body lead to the rightward displacement of the heart tube.

    更新日期:2019-12-18
  • Structural Studies of Overlapping Dinucleosomes in Solution
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Atsushi Matsumoto; Masaaki Sugiyama; Zhenhai Li; Anne Martel; Lionel Porcar; Rintaro Inoue; Daiki Kato; Akihisa Osakabe; Hitoshi Kurumizaka; Hidetoshi Kono

    An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In the present study, the solution structure of the OLDN was investigated through integration of small-angle X-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis, and searched for the conformations that well reproduced the SAXS and SANS scattering curves. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.

    更新日期:2019-12-18
  • Biomechanical Analysis of Angular Motion in Association with Bilateral Semicircular Canal Function
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Shuang Shen; Fei Zhao; Zhaoyue Chen; Shen Yu; Tongtao Cao; Peng Ma; Qingyin Zheng

    The aim of this study was to characterize cupular deformation by calculating the degree of cupular expansion and cupular deflection using a finite element model of bilateral human semicircular canals (SCCs). The results showed that cupular deflection responses were consistent with Ewald’s Ⅱ law, whereas each pair of bilateral cupulae simultaneously expanded or compressed to the same degree. In addition, both the degree of cupular expansion and cupular deflection can be expressed as the solution of forced oscillation during head sinusoidal rotation, and the amplitude of cupular expansion was approximately two times greater than that of cupular deflection. Regarding the amplitude-frequency and phase-frequency characteristics, the amplitude ratios among the horizontal SCC, the anterior SCC and the posterior SCC cupular expansion was constant at 1:0.82:1.62, and the phase differences among them were constant at 0 or 180 degrees at the frequencies of 0.5 to 6 Hz. However, both the amplitude ratio and the phase differencies of the cupular deflection increased nonlinearly with the increase of frequency and tended to be constant at the frequency band between 2 and 6 Hz. The results indicate that the responses of cupular expansion might only be related to the mass and rigidity of three cupulae and the endolymph, but the responses of cupular deflection are related to the mass, rigidity, or damping of them, and these physical properties would be affected by vestibular dysfunction. Therefore, both the degree of cupular expansion and cupular deflection should be considered important mechanical variables for induced neural signals, as these variables provide a better understanding of the SCCs system’s role in the vestibulo-ocular reflex during the clinical rotating chair test and the vestibular autorotation test. Such a numerical model can be further built to provide a useful theoretical approach for exploring the biomechanical nature underlying vestibular dysfunction.

    更新日期:2019-12-18
  • DROIDS 3.0 - detecting genetic and drug class variant impact on conserved protein binding dynamics
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Gregory A. Babbitt; Ernest P. Fokoue; Joshua R. Evans; Kyle I. Diller; Lily E. Adams

    The application of statistical methods to comparatively framed questions about the molecular dynamics (MD) of proteins can potentially enable investigations of biomolecular function beyond the current sequence and structural methods in bioinformatics. However, the chaotic behavior in single MD trajectories requires statistical inference that is derived from large ensembles of simulations representing the comparative functional states of a protein under investigation. Meaningful interpretation of such complex forms of big data poses serious challenges to users of MD. Here, we announce DROIDS 3.0, a method and software package for comparative protein dynamics that includes maxDemon 1.0, a multi-method machine learning application that trains on large ensemble comparisons of concerted protein motions in opposing functional states generated by DROIDS, and deploys learned classifications of these states onto newly generated MD simulations. Local canonical correlations in learning patterns generated from independent, yet identically prepared, MD validation runs are used to identify regions of functionally conserved protein dynamics. The subsequent impacts of genetic and/or drug class variants on conserved dynamics can also be analyzed by deploying the classifiers on variant MD simulations and quantifying how often these altered protein systems display opposing functional states. Here, we present several case studies of complex changes in functional protein dynamics caused by temperature, genetic mutation, and binding interactions with nucleic acids and small molecules. We demonstrate that our machine learning algorithm can properly identify regions of functionally conserved dynamics in ubiquitin and TATA binding protein (TBP). We quantify the impact of genetic variation in TBP and drug class variation targeting the ATP binding region of Hsp90 on conserved dynamics. We identify regions of conserved dynamics in Hsp90 that connect the ATP binding pocket to other functional regions. We also demonstrate that dynamic impacts of various Hsp90 inhibitors rank accordingly with how closely they mimic natural ATP binding.

    更新日期:2019-12-18
  • Rapid simulation of unprocessed DEER decay data for protein fold prediction
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Diego del Alamo; Maxx H. Tessmer; Richard A. Stein; Jimmy B. Feix; Hassane S. Mchaourab; Jens Meiler

    Despite advances in sampling and scoring strategies, Monte Carlo modeling methods still struggle to accurately predict de novo the structures of large proteins, membrane proteins, or proteins of complex topologies. Previous approaches have addressed these shortcomings by leveraging sparse distance data gathered using site-directed spin labeling and electron paramagnetic resonance spectroscopy (SDSL-EPR) to improve protein structure prediction and refinement outcomes. However, existing computational implementations entail compromises between coarse-grained models of the spin label that lower the resolution and explicit models that lead to resource-intense simulations. These methods are further limited by their reliance on distance distributions, which are calculated from a primary refocused echo decay signal and contain uncertainties that may require manual refinement. Here, we addressed these challenges by developing RosettaDEER, a scoring method within the Rosetta software suite capable of simulating DEER distance distributions and decay traces between spin labels fast enough to fold proteins de novo. We demonstrate that the accuracy of resulting distance distributions match or exceed those generated by more computationally intensive methods. Moreover, decay traces generated from these distributions recapitulate intermolecular background coupling parameters, even when the time window of EPR data collection is truncated. As a result, RosettaDEER can discriminate between poorly folded and native-like models using decay traces that cannot be accurately converted into distance distributions using regularized fitting approaches. Finally, using two challenging test cases, we demonstrate that RosettaDEER leverages these experimental data for protein fold prediction more effectively than previous methods. These benchmarking results confirm that RosettaDEER can effectively leverage sparse experimental data for a wide array of modeling applications built into the Rosetta software suite.

    更新日期:2019-12-18
  • Remodeling promotes pro-arrhythmic disruption of calcium homeostasis in failing atrial myocytes
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Y. Shiferaw; G.L. Aistrup; W.E. Louch; J.A. Wasserstrom

    It is well known that heart failure (HF) typically coexists with atrial fibrillation (AF). However, until now, no clear mechanism has been established that relates HF to AF. In this study we apply a multi-scale computational framework to establish a mechanistic link between atrial myocyte structural remodeling in HF and AF. Using a spatially distributed model of calcium (Ca) signaling, we show that disruption of the spatial relationship between L-type Ca channels (LCC) and Ryanodine receptor (RyR) results in markedly increased calcium (Ca) content of the sarcoplasmic reticulum (SR). This increase in SR load is due to changes in the balance between Ca entry via LCC, and Ca extrusion due to the sodium-calcium exchanger, following an altered spatial relationship between these signaling proteins. Next, we show that the increased SR load in atrial myocytes predispose these cells to subcellular Ca waves which occur during the action potential (AP), and are triggered by LCC openings. These waves are common in atrial cells because of the absence of a well-developed t-tubule system in most of these cells. This distinct spatial architecture allows for the presence of a large pool of orphaned RyR receptors, which can fire and sustain Ca waves during the AP. Finally, we incorporate our atrial cell model in two-dimensional tissue simulations, and demonstrate that triggered wave generation in cells leads to electrical waves in tissue which tend to fractionate to form wavelets of excitation. This fractionation is driven by the underlying stochasticity of subcellular Ca waves, which perturbs AP repolarization, and consequently induces localized conduction block in tissue. We outline the mechanism for this effect and argue that it may explain the propensity for atrial arrhythmias in HF.

    更新日期:2019-12-18
  • Mapping multiple distances in a multi-domain protein for the identification of folding intermediates
    Biophys. J. (IF 3.665) Pub Date : 2019-12-18
    Michele Cerminara; Antonie Schöne; Ilona Ritter; Matteo Gabba; Jörg Fitter

    The investigation and understanding of the folding mechanism of multi-domain proteins is still a challenge in structural biology. The use of single molecule Förster resonance energy transfer (smFRET) offers a unique tool to map conformational changes within the proteins structure. Here we present a study following denaturant induced unfolding transitions of yeast phosphoglycerate kinase (yPGK) by mapping several inter- and intra-domain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. One the one hand, the development of the inter-domain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intra-domain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intra-domain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by smFRET measurements. Our results suggest that, in order to fully characterize the complex folding/unfolding mechanism of multi-domain proteins, it is necessary to monitor multiple intra- and inter-domain distances, since a single reporter can lead to a misleading, partial or oversimplified interpretation.

    更新日期:2019-12-18
  • CARDIOMYOPATHY MUTATION ALTERS END-TO-END JUNCTION OF TROPOMYOSIN AND REDUCES CALCIUM SENSITIVITY
    Biophys. J. (IF 3.665) Pub Date : 2019-12-14
    SaiLavanyaa Sundar; Michael J. Rynkiewicz; Anita Ghosh; William Lehman; Jeffrey R. Moore

    Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (Blocked, Closed and Open) in response to calcium-binding to troponin and acto-myosin cross-bridge formation. The Tpm coiled coil polymerizes head-to-tail along the long-pitch helix of F-actin to form continuous super-helical cables that wrap around the actin filaments. The end-to-end bonds formed between the N- and C- terminus of adjacent Tpm molecules define Tpm continuity and play a critical role in the ability of Tpm to cooperatively bind to actin, thus facilitating Tpm conformational switching to cooperatively propagate along F-actin. We expect that a missense mutation in this critical overlap region associated with dilated cardiomyopathy, A277V, will alter tropomyosin binding and thin filament activation by altering the overlap structure. Here, we used co-sedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin, and its ability to regulate acto-myosin interactions. Analytical viscometry coupled with molecular dynamics simulations showed that the A277V mutation results in enhanced Tpm end-to-end bond strength and a reduced curvature of the Tpm overlap domain. The mutant Tpm exhibited enhanced actin - Tpm binding affinity, consistent with overlap stabilization. The observed A277V-induced decrease in cooperative activation observed with regulated thin filament motility indicates that increased overlap stabilization is not correlated with Tpm-Tpm overlap binding strength or mechanical rigidity as is often assumed. Instead, A277V-induced structural changes result in local and delocalized increases in tropomyosin flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca2+ sensitivity, consistent with a mutation-induced bolstering of B-state Tpm-actin electrostatic contacts and an increased TnT1 binding affinity, was also observed.

    更新日期:2019-12-17
  • Physiochemical modelling of vesicle dynamics upon osmotic upshift
    Biophys. J. (IF 3.665) Pub Date : 2019-12-14
    Matteo Gabba; Bert Poolman

    We modelled the relaxation dynamics of (lipid) vesicles upon osmotic upshift taking into account volume variation, chemical reaction kinetics and passive transport across the membrane. We focused on the relaxation-kinetics upon addition of impermeable osmolytes like KCl and membrane-permeable solutes like weak acids. We studied the effect of the most relevant physical parameters on the dynamic behavior of the system, as well as, on the relaxation rates. We observe that: (i) the dynamic complexity of the relaxation kinetics depends on the number of permeable species; (ii) the permeability coefficients (P) and the weak acid strength (PKa’s) determine the dynamic behavior of the system; (iii) the vesicle size does not affect the dynamics but only the relaxation rates of the system; and (iv) heterogeneities in the vesicle size provoke stretching of the relaxation curves. The model was successfully benchmarked for determining permeability coefficients by fitting of our experimental relaxation curves and by comparison of the data with literature values (see page XX of the same issue). To describe the dynamics of yeast cells upon osmotic upshift, we extended the model to account for turgor pressure and non-osmotic volume. Significance statement Physiochemical kinetic models are an important set of tools for the understanding of biochemical and biological systems. We present a comprehensive description of the relaxation dynamics of vesicles upon osmotic shock which includes volume variation, reaction kinetics, passive permeability across the membrane, and turgor pressure. The model is a flexible platform for the description of many biochemical systems. Owing to its generality, the model is easily extended with the addition of new features for the interpretation of in vitro experiments with protein transporters, electrochemical studies and uptake experiments.

    更新日期:2019-12-17
  • Ligand Entry into Fatty Acid Binding Protein via Local Unfolding instead of Gap Widening
    Biophys. J. (IF 3.665) Pub Date : 2019-12-14
    Tianshu Xiao; Yimei Lu; Jing-song Fan; Daiwen Yang

    Fatty acid binding proteins (FABPs) play an important role in transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal FABP was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still takes up fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like state, and two locally unfolded states in aqueous solution. Local unfolding of either βB–βD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process.

    更新日期:2019-12-17
  • Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision using a Zigzag Force Ramp
    Biophys. J. (IF 3.665) Pub Date : 2019-12-13
    David R. Jacobson; Lyle Uyetake; Thomas T. Perkins

    Precise quantification of the energetics and interactions that stabilize membrane proteins in a lipid bilayer is a long-sought goal. Toward this end, atomic force microscopy (AFM) has been used to unfold individual membrane proteins embedded in their native lipid bilayer, typically by retracting the cantilever at a constant velocity. Recently, unfolding intermediates separated by as few as two amino acids were detected using focused-ion-beam modified ultrashort cantilevers. However, unambiguously discriminating between such closely spaced states remains challenging, in part, because any individual unfolding trajectory only occupies a subset of the total number of intermediates. Moreover, structural assignment of these intermediates via worm-like-chain analysis is hindered by brief dwell times compounded with thermal and instrumental noise. To overcome these issues, we moved the cantilever in a sawtooth pattern of 6–12 nm, offset by 0.25–1 nm per cycle, generating a “zigzag” force ramp of alternating positive and negative loading rates. We applied this protocol to the model membrane protein bacteriorhodopsin (bR). In contrast to conventional studies that extract bR’s photoactive retinal along with the first transmembrane helix, we unfolded bR in the presence of its retinal. To do so, we introduced a previously developed enzymatic-cleavage site between helices E and F and pulled from the top of the E helix using a site-specific, covalent attachment. The resulting zigzag unfolding trajectories occupied 40% more states per trajectory and occupied those states for longer times than traditional constant-velocity records. In total, we identified 31 intermediates during the unfolding of five helices of EF-cleaved bR. These included a previously reported, mechanically robust intermediate located between helices C and B that with our enhanced resolution is now shown to be two distinct states separated by three amino acids. Interestingly, another intermediate directly interacted with the retinal, an interaction confirmed by removing the retinal.

    更新日期:2019-12-13
  • The Auditory Mechanics of the Outer Ear of the Bush-Cricket: A Numerical Approach
    Biophys. J. (IF 3.665) Pub Date : 2019-12-12
    Emine Celiker; Thorin Jonsson; Fernando Montealegre-Z

    Bush-crickets have tympanal ears located in the forelegs. Their ears are elaborated as they have outer, middle and inner ear components. The outer ear comprises an air-filled tube derived from the respiratory trachea, the acoustic trachea (AT), which transfers sound from the prothoracic acoustic spiracle to the internal side of the ear drums in the legs. A key feature of the AT is its capacity to reduce the velocity of sound propagation and alter the acoustic driving forces of the tympanum (the ear drum), producing differences in sound pressure and time between the left and right sides, therefore aiding the directional hearing of the animal. It has been demonstrated experimentally that the tracheal sound transmission generates a gain of approximately 15 dB and a propagation velocity of 255 ms-1, an approximately 25% reduction from free-field propagation. However the mechanism responsible for this change in sound pressure level and velocity remains elusive. In this study, we investigate the mechanical processes behind the sound pressure gain in the AT by numerically modelling the tracheal acoustic behaviour using the finite element method and real 3D geometries of the tracheae of the bush-cricket Copiphora gorgonensis. Taking into account the thermoviscous acoustic-shell interaction on the propagation of sound, we analyse the effects of the horn-shaped domain, material properties of the tracheal wall and the thermal processes on the change in sound pressure level in the AT. Through the numerical results obtained it is discerned that the tracheal geometry is the main factor contributing to the observed pressure gain.

    更新日期:2019-12-13
  • Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence
    Biophys. J. (IF 3.665) Pub Date : 2019-12-12
    Isaac Angert; Siddarth Reddy Karuka; Jared Hennen; Yan Chen; Joseph P. Albanesi; Louis M. Mansky; Joachim D. Mueller

    Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions including cell motility, signal transduction, and virus replication. Here we describe a dual color (DC) extension of the fluorescence z-scan technique which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the co-expression of a second distinctly colored fluorescent protein provides a soluble reference species, which delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ∼16 μM and Hill coefficient of n ∼2.

    更新日期:2019-12-13
  • Modulation of kinesin’s load-bearing capacity by force geometry and the microtubule track
    Biophys. J. (IF 3.665) Pub Date : 2019-12-12
    Serapion Pyrpassopoulos; Henry Shuman; E. Michael Ostap

    Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule associated proteins and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long-axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration < 0.2 s) to a persistent motor that sustains attachment (median attachment duration > 3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice which has implications for a range of cellular activities including cell division and organelle transport.

    更新日期:2019-12-13
  • Ensembles of breathing nucleosomes: a computational study
    Biophys. J. (IF 3.665) Pub Date : 2019-12-12
    Koen van Deelen; Helmut Schiessel; Lennart de Bruin

    About 3/4 of the human DNA molecules are wrapped into nucleosomes, protein spools with DNA. Nucleosomes are highly dynamic, transiently exposing their DNA through spontaneous unspooling. Recent experiments allowed to observe the DNA of an ensemble of such breathing nucleosomes through x-ray diffraction with contrast matching between the solvent and the protein core. In the current study we calculate such an ensemble through a Monte Carlo simulation of a coarse-grained nucleosome model with sequence-dependent DNA mechanics. Our analysis gives detailed insights into the sequence-dependence of nucleosome breathing observed in the experiment and allows to determine the adsorption energy of the DNA bound to the protein core as a function of the ionic strength. Moreover, we predict the breathing behaviour of other potentially interesting sequences and compare the findings to earlier related experiments.

    更新日期:2019-12-13
  • Structure of an unfolding intermediate of a RRM domain of ETR-3 protein reveals its native-like fold
    Biophys. J. (IF 3.665) Pub Date : 2019-12-06
    Harshesh Bhatt, Akshay Kumar Ganguly, Sonam Sharma, Gajraj Singh Kushwaha, Mohammad Firoz Khan, Sobhan Sen, Neel Sarovar Bhavesh

    Prevalence of one or more partially folded intermediates during protein unfolding with different secondary and ternary conformations has been identified as an integral character of protein unfolding. These transition state species need to be characterized structurally for elucidation of its folding pathways. We have determined the three-dimensional structure of an intermediate state with increased conformational space sampling under urea denaturing condition. The protein unfolds completely at 10 M urea but retains residual secondary structural propensities with restricted motion. Here we describe the native state, observable intermediate state and unfolded state for ETR-3 RRM-3, which has canonical RRM fold. These observations can shed more light on unfolding events for RRM containing proteins. Statement of significance The study presents solution structure and dynamics of native, an unfolding intermediate and explores the structural propensities in unfolded state of a RNA Recognition Motif of ETR-3 protein. The delineation of unfolding pathway at atomic-resolution of a canonical RNA recognition motif is likely to help in understanding the unfolding events in other RRMs involved in disease causing conditions upon misfolding.

    更新日期:2019-12-06
  • The Reactions of O2 and NO with Mixed-Valence ba3 Cytochrome c Oxidase from Thermus thermophilus
    Biophys. J. (IF 3.665) Pub Date : 2019-12-06
    I. Szundi, C. Funatogawa, T. Soulimani, Ó. Einarsdóttir

    Earlier CO-flow-flash experiments on the fully reduced Thermus thermophilus ba3 (Tt ba3) cytochrome oxidase revealed that O2 binding was slowed down by a factor of 10 in the presence of carbon monoxide (CO) (Szundi et al., 2010, PNAS 107, 21010-21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the CuB+-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba3 (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380-1390) interferes with O2 and NO binding and the ability of CuB to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba3 with O2 following the photodissociation of CO were investigated using time-resolved optical absorption spectroscopy using photolabile O2 and nitric oxide (NO) carriers. No electron backflow was detected following photolysis of the mixed-valence CO-bound Tt ba3. The rate of O2 and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived CuB+-CO complex did not prevent O-O bond splitting and the resulting PM formation, which was significantly faster (5-10 times) than in the bovine heart enzyme. We propose that O2 binding to heme a3 in Tt ba3 causes CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects, thus allowing CuB+ to act as an electron donor in the mixed-valence enzyme. The significantly faster O2 binding and O-O bond cleavage in Tt ba3 compared to analogous steps in the aa3 oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.

    更新日期:2019-12-06
  • PSI-SMALP, a detergent-free cyanobacterial photosystem I reveals faster femtosecond photochemistry
    Biophys. J. (IF 3.665) Pub Date : 2019-12-06
    Dmitry A. Cherepanov, Nathan G. Brady, Ivan V. Shelaev, Jon Nguyen, Fedor E. Gostev, Mahir D. Mamedov, Victor A. Nadtochenko, Barry D. Bruce

    Cyanobacterial Photosystem I (PSI) functions as a light-driven cyt c6-ferredoxin/oxidoreductase located in the thylakoid membrane. In the present work, the energy- and charge- transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-β-D-maltoside solubilization (DM-PSI) and a new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll (LWC) forms within 0.1–20 ps and revealed little or no charge separation and oxidation of the special pair, P700. The formation of ion-radical pair P700+A1− occurred with a characteristic time of 36 ps, being kinetically controlled by energy transfer from the LWC to P700. Quite surprisingly, the detergent-free SMA-PSI complexes upon excitation by these long-wave pulses undergo an ultra-fast (<100 fs) charge separation in ∼45% of particles. In the remaining complexes (∼55%) the energy transfer to P700 occurred at ∼36 ps similar to the DM-PSI. Both isolation methods result in a trimeric form of PSI, yet the SMA-PSI complexes display a heterogenous kinetic behavior. The much faster rate of charge separation suggests the existence of an ultrafast pathway for charge separation in the SMA-PSI that may be disrupted during detergent isolation.

    更新日期:2019-12-06
  • Docking Troponin-T onto the Tropomyosin Overlapping Domain of Thin Filaments
    Biophys. J. (IF 3.665) Pub Date : 2019-12-06
    E. Pavadai, M.J. Rynkiewicz, A. Ghosh, W. Lehman

    Complete description of thin filament conformational transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. To date, several molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin because high-resolution experimentally determined structures of filament-associated troponin are not available. However, previously published all-atom models of the thin filament show chain separation and corruption of components during our molecular dynamics simulations of the models, implying artefactual subunit organization, possibly due to incorporation of unorthodox tropomyosin-TnT crystal structures and complex FRET measurements during model construction. For example, the recent Williams et al. (2016) atomistic model of the thin filament displays a paucity of salt bridges and hydrophobic complementarity between the TnT-tail (TnT1) and tropomyosin, which is difficult to reconcile with the high, 20 nM Kd binding of TnT onto tropomyosin. Indeed, our molecular dynamics simulations show the TnT1 component in their model partially dissociates from tropomyosin in under 100 ns, while actin-tropomyosin and TnT1 models themselves remain intact. We therefore revisited computational work aiming to improve TnT1-thin filament models by employing unbiased docking methodologies, which test billions of trial rotations and translations of TnT1 over three-dimensional grids covering end-to-end bonded tropomyosin alone or tropomyosin on F-actin. We limited conformational searches to the association of well-characterized TnT1 helical-domains and either isolated tropomyosin or actin-tropomyosin, yet avoided docking TnT domains that lack known or predicted structure. The docking programs PIPER and ClusPro were used, followed by interaction energy optimization and extensive molecular dynamics. TnT1 docked to either side of isolated tropomyosin, but uniquely onto one location of actin-bound tropomyosin. The anti-parallel interaction with tropomyosin contained abundant salt bridges and intimately integrated hydrophobic networks joining TnT1 and the tropomyosin N-/C-terminal overlapping-domain. The TnT1-tropomyosin linkage yields well-defined molecular crevices. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previously proposed models.

    更新日期:2019-12-06
  • Biophysics and the Genomic Sciences
    Biophys. J. (IF 3.665) Pub Date : 2019-07-30
    David C. Schwartz

    It is now rare to find biological, or genetic investigations that do not rely on the tools, data, and thinking drawn from the genomic sciences. Much of this revolution is powered by contemporary sequencing approaches that readily deliver large, genome-wide data sets that not only provide genetic insights but also uniquely report molecular outcomes from experiments that biophysicists are increasingly using for potentiating structural and mechanistic investigations. In this perspective, I describe a path of how biophysical thinking greatly contributed to this revolution in ways that parallel advancements in computer science through discussion of several key inventions, described as “foundational devices.” These discussions also point at the future of how biophysics and the genomic sciences may become more finely integrated for empowering new measurement paradigms for biological investigations.

    更新日期:2019-12-03
  • Structural Basis of Enhanced Facilitated Diffusion of DNA Binding Proteins in Crowded Cellular Milieu
    Biophys. J. (IF 3.665) Pub Date : 2019-11-29
    Pinki Dey, Arnab Bhattacherjee

    While the fast association between DBPs and DNA is explained by a facilitated diffusion mechanism, where DBPs adopt a weighted combination of 3D diffusion and 1D sliding and hopping modes of transportation, the role of cellular environment that contains many nonspecifically interacting proteins and other biomolecules is mostly overlooked. By performing large scale computational simulations with an appropriately tuned model of protein and DNA in the presence of nonspecifically interacting bulk and DNA bound crowders (genomic crowders), we demonstrate the structural basis of the enhanced facilitated diffusion of DBPs inside a crowded cellular milieu through novel 1D scanning mechanisms. In the presence of bulk crowders, we identify the protein to float along the DNA under the influence of protein-crowder nonspecific interactions. The search mode is distinctly different compared to usual 1D sliding and hopping dynamics where protein diffusion is regulated by the DNA electrostatics. In contrast, the presence of genomic crowders expedites the target search process by transporting the protein over DNA segments through the formation of a transient protein-crowder bridged complex. By analyzing the ruggedness of the associated potential energy landscape, we underpin the molecular origin of the kinetic advantages of these search modes and show that they successfully explain the experimentally observed acceleration of facilitated diffusion of DBPs by molecular crowding agents and crowder concentration-dependent enzymatic activity of transcription factors. Our findings provide crucial insights into gene regulation kinetics inside the crowded cellular milieu.

    更新日期:2019-11-29
  • Area and Geometry Dependence of Cell Migration in Asymmetric Two-State Micropatterns
    Biophys. J. (IF 3.665) Pub Date : 2019-11-29
    Alexandra Fink, David B. Brückner, Christoph Schreiber, Peter J.F. Röttgermann, Chase P. Broedersz, Joachim O. Rädler

    Micro-structured surfaces provide a unique framework to probe cell migration and cytoskeletal dynamics in a standardized manner. Here, we report on the steady-state occupancy probability of cells in asymmetric two-state microstructures that consist of two fibronectin-coated adhesion sites connected by a thin guidance cue. In these dumbbell-like structures, cells transition between the two sites in a repeated and stochastic manner and average dwell times in the respective microenvironments are determined from the cell trajectories. We study the dynamics of human breast carcinoma cells (MDA-MB-231) in these microstructures as a function of area, shape and orientation of the adhesion sites. On square adhesive sites with different areas, we find that the occupancy probability ratio is directly proportional to the ratio of corresponding adhesion site areas. These asymmetries are well captured by a simple model for the stochastic nonlinear dynamics of the cells which reveals generic features of the motion. Sites of equal area but different shape lead to equal occupancy, if shapes are isotropic, e.g. squared or circular. In contrast, an asymmetry in the occupancy is induced by anisotropic shapes like rhombi, triangles or rectangles that enable motion in the direction perpendicular to the transition axis. Analysis of the 2D motion of cells between two rectangles with orthogonal orientation suggests that cellular transition rates depend on the cell polarisation induced by anisotropic micropatterns. Taken together, our results illustrate how two-state-micropatterns provide a dynamic migration assay with distinct dwell times and relative cell occupancy as readouts, which may be useful to probe cell-microenvironment interactions.

    更新日期:2019-11-29
  • Folding of the β-barrel membrane protein OmpA into nanodiscs
    Biophys. J. (IF 3.665) Pub Date : 2019-11-28
    DeeAnn.K. Asamoto, Guipeun Kang, Judy E. Kim

    Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely-diffusing NDs. In this report, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopy to confirm the spontaneous folding of Outer Membrane Protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The CD spectra of OmpA in NDs and SUVs were similar, and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by UVRR, which revealed the intense 785 cm-1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was 2- to 3-fold slower in NDs compared to in SUVs, and this decreased rate can be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments, and offer benefits of optical clarity, sample homogeneity, control of ND-to-protein ratios, and greater stability.

    更新日期:2019-11-28
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