Biophysical Journal

Effect of Antenna-Depletion in Photosystem II on Excitation Energy Transfer in Arabidopsis thaliana.

Publication Date: 2010 Mar 3 PMID: 20197046
Authors: van Oort, B. - Alberts, M. - de Bianchi, S. - Dall'osto, L. - Bassi, R. - Trinkunas, G. - Croce, R. - van Amerongen, H.
Journal: Biophys J

The role of individual photosynthetic antenna complexes of Photosystem II (PSII) both in membrane organization and excitation energy transfer have been investigated. Thylakoid membranes from wild-type Arabidopsis thaliana, and three mutants lacking light-harvesting complexes CP24, CP26, or CP29, respectively, were studied by picosecond-fluorescence spectroscopy. By using different excitation/detection wavelength combinations it was possible for the first time, to our knowledge, to separate PSI and PSII fluorescence kinetics. The sub-100 ps component, previously ascribed entirely to PSI, turns out to be due partly to PSII. Moreover, the migration time of excitations from antenna to PSII reaction center (RC) was determined for the first time, to our knowledge, for thylakoid membranes. It is four times longer than for PSII-only membranes, due to additional antenna complexes, which are less well connected to the RC. The results in the absence of CP26 are very similar to those of wild-type, demonstrating that the PSII organization is not disturbed. However, the kinetics in the absence of CP29 and, especially, of CP24 show that a large fraction of the light-harvesting complexes becomes badly connected to the RCs. Interestingly, the excited-state lifetimes of the disconnected light-harvesting complexes seem to be substantially quenched.

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The Epidermal Ca(2+) Gradient: Measurement Using the Phasor Representation of Fluorescent Lifetime Imaging.

Publication Date: 2010 Mar 3 PMID: 20197045
Authors: Celli, A. - Sanchez, S. - Behne, M. - Hazlett, T. - Gratton, E. - Mauro, T.
Journal: Biophys J

Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.

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Accounting for Ligand Conformational Restriction in Calculations of Protein-Ligand Binding Affinities.

Publication Date: 2010 Mar 3 PMID: 20197044
Authors: Gao, C. - Park, M. S. - Stern, H. A.
Journal: Biophys J

The conformation adopted by a ligand on binding to a receptor may differ from its lowest-energy conformation in solution. In addition, the bound ligand is more conformationally restricted, which is associated with a configurational entropy loss. The free energy change due to these effects is often neglected or treated crudely in current models for predicting binding affinity. We present a method for estimating this contribution, based on perturbation theory using the quasi-harmonic model of Karplus and Kushick as a reference system. The consistency of the method is checked for small model systems. Subsequently we use the method, along with an estimate for the enthalpic contribution due to ligand-receptor interactions, to calculate relative binding affinities. The AMBER force field and generalized Born implicit solvent model is used. Binding affinities were estimated for a test set of 233 protein-ligand complexes for which crystal structures and measured binding affinities are available. In most cases, the ligand conformation in the bound state was significantly different from the most favorable conformation in solution. In general, the correlation between measured and calculated ligand binding affinities including the free energy change due to ligand conformational change is comparable to or slightly better than that obtained by using an empirically-trained docking score. Both entropic and enthalpic contributions to this free energy change are significant.

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Universality in Protein Residue Networks.

Publication Date: 2010 Mar 3 PMID: 20197043
Authors: Estrada, E.
Journal: Biophys J

Residue networks representing 595 nonhomologous proteins are studied. These networks exhibit universal topological characteristics as they belong to the topological class of modular networks formed by several highly interconnected clusters separated by topological cavities. There are some networks that tend to deviate from this universality. These networks represent small-size proteins having <200 residues. This article explains such differences in terms of the domain structure of these proteins. On the other hand, the topological cavities characterizing proteins residue networks match very well with protein binding sites. This study investigates the effect of the cutoff value used in building the residue network. For small cutoff values, <5 A, the cavities found are very large corresponding almost to the whole protein surface. On the contrary, for large cutoff value, >10.0 A, only very large cavities are detected and the networks look very homogeneous. These findings are useful for practical purposes as well as for identifying protein-like complex networks. Finally, this article shows that the main topological class of residue networks is not reproduced by random networks growing according to Erdos-Renyi model or the preferential attachment method of Barabasi-Albert. However, the Watts-Strogatz model reproduces very well the topological class as well as other topological properties of residue network. A more biologically appealing modification of the Watts-Strogatz model to describe residue networks is proposed.

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Left-Handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity among Receptor Tyrosine Kinases.

Publication Date: 2010 Mar 3 PMID: 20197042
Authors: Bocharov, E. V. - Mayzel, M. L. - Volynsky, P. E. - Mineev, K. S. - Tkach, E. N. - Ermolyuk, Y. S. - Schulga, A. A. - Efremov, R. G. - Arseniev, A. S.
Journal: Biophys J

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.

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Statistics and Physical Origins of pK and Ionization State Changes upon Protein-Ligand Binding.

Publication Date: 2010 Mar 3 PMID: 20197041
Authors: Aguilar, B. - Anandakrishnan, R. - Ruscio, J. Z. - Onufriev, A. V.
Journal: Biophys J

This work investigates statistical prevalence and overall physical origins of changes in charge states of receptor proteins upon ligand binding. These changes are explored as a function of the ligand type (small molecule, protein, and nucleic acid), and distance from the binding region. Standard continuum solvent methodology is used to compute, on an equal footing, pK changes upon ligand binding for a total of 5899 ionizable residues in 20 protein-protein, 20 protein-small molecule, and 20 protein-nucleic acid high-resolution complexes. The size of the data set combined with an extensive error and sensitivity analysis allows us to make statistically justified and conservative conclusions: in 60% of all protein-small molecule, 90% of all protein-protein, and 85% of all protein-nucleic acid complexes there exists at least one ionizable residue that changes its charge state upon ligand binding at physiological conditions (pH = 6.5). Considering the most biologically relevant pH range of 4-8, the number of ionizable residues that experience substantial pK changes (DeltapK > 1.0) due to ligand binding is appreciable: on average, 6% of all ionizable residues in protein-small molecule complexes, 9% in protein-protein, and 12% in protein-nucleic acid complexes experience a substantial pK change upon ligand binding. These changes are safely above the statistical false-positive noise level. Most of the changes occur in the immediate binding interface region, where approximately one out of five ionizable residues experiences substantial pK change regardless of the ligand type. However, the physical origins of the change differ between the types: in protein-nucleic acid complexes, the pK values of interface residues are predominantly affected by electrostatic effects, whereas in protein-protein and protein-small molecule complexes, structural changes due to the induced-fit effect play an equally important role. In protein-protein and protein-nucleic acid complexes, there is a statistically significant number of substantial pK perturbations, mostly due to the induced-fit structural changes, in regions far from the binding interface.

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Determination of Ensemble-Average Pairwise Root Mean-Square Deviation from Experimental B-Factors.

Publication Date: 2010 Mar 3 PMID: 20197040
Authors: Kuzmanic, A. - Zagrovic, B.
Journal: Biophys J

Root mean-square deviation (RMSD) after roto-translational least-squares fitting is a measure of global structural similarity of macromolecules used commonly. On the other hand, experimental x-ray B-factors are used frequently to study local structural heterogeneity and dynamics in macromolecules by providing direct information about root mean-square fluctuations (RMSF) that can also be calculated from molecular dynamics simulations. We provide a mathematical derivation showing that, given a set of conservative assumptions, a root mean-square ensemble-average of an all-against-all distribution of pairwise RMSD for a single molecular species, (1/2), is directly related to average B-factors () and (1/2). We show this relationship and explore its limits of validity on a heterogeneous ensemble of structures taken from molecular dynamics simulations of villin headpiece generated using distributed-computing techniques and the Folding@Home cluster. Our results provide a basis for quantifying global structural diversity of macromolecules in crystals directly from x-ray experiments, and we show this on a large set of structures taken from the Protein Data Bank. In particular, we show that the ensemble-average pairwise backbone RMSD for a microscopic ensemble underlying a typical protein x-ray structure is approximately 1.1 A, under the assumption that the principal contribution to experimental B-factors is conformational variability.

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Probing the DNA-Binding Affinity and Specificity of Designed Zinc Finger Proteins.
Publication Date: 2010 Mar 3 PMID: 20197039
Authors: Jantz, D. - Berg, J. M.
Journal: Biophys J

Engineered transcription factors and endonucleases based on designed Cys(2)His(2) zinc finger domains have proven to be effective tools for the directed regulation and modification of genes. The introduction of this technology into both research and clinical settings necessitates the development of rapid and accurate means of evaluating both the binding affinity and binding specificity of designed zinc finger domains. Using a fluorescence anisotropy-based DNA-binding assay, we examined the DNA-binding properties of two engineered zinc finger proteins that differ by a single amino acid. We demonstrate that the protein with the highest affinity for a particular DNA site need not be the protein that binds that site with the highest degree of specificity. Moreover, by comparing the binding characteristics of the two proteins at varying salt concentrations, we show that the ionic strength makes significant and variable contributions to both affinity and specificity. These results have significant implications for zinc finger design as they highlight the importance of considering affinity, specificity, and environmental requirements in designing a DNA-binding domain for a particular application.

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The Interaction of alphaB-Crystallin with Mature alpha-Synuclein Amyloid Fibrils Inhibits Their Elongation.
Publication Date: 2010 Mar 3 PMID: 20197038
Authors: Waudby, C. A. - Knowles, T. P. - Devlin, G. L. - Skepper, J. N. - Ecroyd, H. - Carver, J. A. - Welland, M. E. - Christodoulou, J. - Dobson, C. M. - Meehan, S.
Journal: Biophys J

alphaB-Crystallin is a small heat-shock protein (sHsp) that is colocalized with alpha-synuclein (alphaSyn) in Lewy bodies-the pathological hallmarks of Parkinson's disease-and is an inhibitor of alphaSyn amyloid fibril formation in an ATP-independent manner in vitro. We have investigated the mechanism underlying the inhibitory action of sHsps, and here we establish, by means of a variety of biophysical techniques including immunogold labeling and nuclear magnetic resonance spectroscopy, that alphaB-crystallin interacts with alphaSyn, binding along the length of mature amyloid fibrils. By measurement of seeded fibril elongation kinetics, both in solution and on a surface using a quartz crystal microbalance, this binding is shown to strongly inhibit further growth of the fibrils. The binding is also demonstrated to shift the monomer-fibril equilibrium in favor of dissociation. We believe that this mechanism, by which a sHsp interacts with mature amyloid fibrils, could represent an additional and potentially generic means by which at least some chaperones protect against amyloid aggregation and limit the onset of misfolding diseases.

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Microscopic Basis for the Mesoscopic Extensibility of Dendrimer-Compacted DNA.
Publication Date: 2010 Mar 3 PMID: 20197037
Authors: Mills, M. - Orr, B. - Banaszak Holl, M. M. - Andricioaei, I.
Journal: Biophys J

The mechanism of DNA compaction by dendrimers is key to the design of nanotechnologies that can deliver genetic material into cells. We present atomistic simulations, mesoscopic modeling and single-molecule pulling experiments describing DNA dendrimer interactions. All-atom molecular dynamics were used to characterize pulling-force-dependent interactions between DNA and generation-3 PAMAM amine-terminated dendrimers, and a free energy profile and mean forces along the interaction coordinate are calculated. The energy, force, and geometry parameters computed at the atomic level are input for a Monte Carlo model yielding mesoscopic force-extension curves. Actual experimental single-molecule curves obtained with optical tweezers are also presented, and they show remarkable agreement with the virtual curves from our model. The calculations reveal the microscopic origin of the hysteresis observed in the phase transition underlying compaction. A broad range of ionic and pulling parameters is sampled, and suggestions for windows of conditions to probe new single-molecule behavior are made.

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Transcription within Condensed Chromatin: Steric Hindrance Facilitates Elongation.
Publication Date: 2010 Mar 3 PMID: 20197036
Authors: Becavin, C. - Barbi, M. - Victor, J. M. - Lesne, A.
Journal: Biophys J

During eukaryotic transcription, RNA-polymerase activity generates torsional stress in DNA, having a negative impact on the elongation process. Using our previous studies of chromatin fiber structure and conformational transitions, we suggest that this torsional stress can be alleviated, thanks to a tradeoff between the fiber twist and nucleosome conformational transitions into an activated state named "reversome". Our model enlightens the origin of polymerase pauses, and leads to the counterintuitive conclusion that chromatin-organized compaction might facilitate polymerase progression. Indeed, in a compact and well-structured chromatin loop, steric hindrance between nucleosomes enforces sequential transitions, thus ensuring that the polymerase always meets a permissive nucleosomal state.

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Peptide-Induced Domain Formation in Supported Lipid Bilayers: Direct Evidence by Combined Atomic Force and Polarized Total Internal Reflection Fluorescence Microscopy.
Publication Date: 2010 Mar 3 PMID: 20197035
Authors: Oreopoulos, J. - Epand, R. F. - Epand, R. M. - Yip, C. M.
Journal: Biophys J

Direct visualization of the mechanism(s) by which peptides induce localized changes to the structure of membranes has high potential for enabling understanding of the structure-function relationship in antimicrobial and cell-penetrating peptides. We have applied a combined imaging strategy to track the interaction of a model antimicrobial peptide, PFWRIRIRR-amide, with bacterial membrane-mimetic supported phospholipid bilayers comprised of POPE/TOCL. Our in situ studies revealed rapid reorganization of the POPE/TOCL membrane into localized TOCL-rich domains with a concomitant change in the organization of the membranes themselves, as reflected by changes in fluorescent-membrane-probe order parameter, upon introduction of the peptide.

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Effect of Membrane Thickness on Conformational Sampling of Phospholamban from Computer Simulations.
Publication Date: 2010 Mar 3 PMID: 20197034
Authors: Sayadi, M. - Tanizaki, S. - Feig, M.
Journal: Biophys J

The conformational sampling of monomeric, membrane-bound phospholamban is described from computer simulations. Phospholamban (PLB) plays a key role as a regulator of sarcoplasmic reticulum calcium ATPase. An implicit membrane model is used in conjunction with replica exchange molecular dynamics simulations to reach mus-ms timescales. The implicit membrane model was also used to study the effect of different membrane thicknesses by scaling the low-dielectric region. The conformational sampling with the membrane model mimicking dipalmitoylphosphatidylcholine bilayers is in good agreement overall with experimental measurements, but consists of a wide variety of different conformations including structures not described previously. The conformational ensemble shifts significantly in the presence of thinner or thicker membranes. This has implications for the structure and dynamics of PLB in physiological membranes and offers what we believe to be a new interpretation of previous experimental measurements of PLB in detergents and microsomal membrane.

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On the Role of Acylation of Transmembrane Proteins.
Publication Date: 2010 Mar 3 PMID: 20197033
Authors: Morozova, D. - Weiss, M.
Journal: Biophys J

Acylation is a frequent means to ensure membrane association of a variety of soluble proteins in living cells. However, many transmembrane proteins are palmitoylated, indicating that this posttranslational modification may also serve as a means to regulate protein trafficking. Based on coarse-grained membrane simulations, we find that protein acylation significantly alters the tilting of transmembrane proteins with respect to the bilayer normal. In addition, the proteins' partitioning behavior and cluster formation ability due to hydrophobic mismatching is strongly altered. Based on our results, we propose that acylation is a potent means to regulate the trafficking of transmembrane proteins along the early secretory pathway.

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Mouse Fibroblast Cell Adhesion Studied by Neutron Reflectometry.
Publication Date: 2010 Mar 3 PMID: 20197032
Authors: Smith, H. L. - Hickey, J. - Jablin, M. S. - Trujillo, A. - Freyer, J. P. - Majewski, J.
Journal: Biophys J

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be approximately 180 A. The membrane region ( approximately 80 A thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.

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An Iris-Like Mechanism of Pore Dilation in the CorA Magnesium Transport System.
Publication Date: 2010 Mar 3 PMID: 20197031
Authors: Chakrabarti, N. - Neale, C. - Payandeh, J. - Pai, E. F. - Pomes, R.
Journal: Biophys J

Magnesium translocation across cell membranes is essential for numerous physiological processes. Three recently reported crystal structures of the CorA magnesium transport system revealed a surprising architecture, with a bundle of giant alpha-helices forming a 60-A-long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. The presence of divalent cations in putative intracellular regulation sites suggests that these structures correspond to the closed conformation of CorA. To examine the nature of the conduction pathway, we performed 110-ns molecular-dynamics simulations of two of these structures in a lipid bilayer with and without regulatory ions. The results show that a 15-A-long hydrophobic constriction straddling the membrane-cytosol interface constitutes a steric bottleneck whose location coincides with an electrostatic barrier opposing cation translocation. In one of the simulations, structural relaxation after the removal of regulatory ions led to concerted changes in the tilt of the pore helices, resulting in iris-like dilation and spontaneous hydration of the hydrophobic neck. This simple and robust mechanism is consistent with the regulation of pore opening by intracellular magnesium concentration, and explains the unusual architecture of CorA.

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Agonist-Induced Changes in Ca(2+) Permeation through the Nociceptor Cation Channel TRPA1.
Publication Date: 2010 Mar 3 PMID: 20197030
Authors: Karashima, Y. - Prenen, J. - Talavera, K. - Janssens, A. - Voets, T. - Nilius, B.
Journal: Biophys J

The Ca(2+)-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca(2+) ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of approximately 11 A. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca(2+), and at a membrane potential of -80 mV, approximately 17% of the inward TRPA1 current is carried by Ca(2+). Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 A and an increase in divalent cation selectivity and fractional Ca(2+) current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca(2+) current also prevented mustard-oil-induced increases in Ca(2+) permeation. It is interesting that fractional Ca(2+) currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca(2+) in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca(2+) influx.

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Modulation of KvAP Unitary Conductance and Gating by 1-Alkanols and Other Surface Active Agents.
Publication Date: 2010 Mar 3 PMID: 20197029
Authors: Finol-Urdaneta, R. K. - McArthur, J. R. - Juranka, P. F. - French, R. J. - Morris, C. E.
Journal: Biophys J

The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.

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Two Open States with Progressive Proton Selectivities in the Branched Channelrhodopsin-2 Photocycle.
Publication Date: 2010 Mar 3 PMID: 20197028
Authors: Berndt, A. - Prigge, M. - Gradmann, D. - Hegemann, P.
Journal: Biophys J

Channelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H(+). Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H(+) and divalent cations (Ca(2+) and/or Mg(2+)), as compared to monovalent cations (Na(+) and/or K(+)). Compared to monovalent cation conductance, the H(+) conductance, alpha, is approximately 3 x 10(6) and the divalent cation conductance, beta, is approximately 0.01 in the early conducting state. In the stationary mixture of the early and late states, alpha is larger and beta smaller, both by a factor of approximately 2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures.

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Harmonic Oscillations in Homeostatic Controllers: Dynamics of the p53 Regulatory System.
Publication Date: 2010 Mar 3 PMID: 20197027
Authors: Jolma, I. W. - Ni, X. Y. - Rensing, L. - Ruoff, P.
Journal: Biophys J

Homeostatic mechanisms are essential for the protection and adaptation of organisms in a changing and challenging environment. Previously, we have described molecular mechanisms that lead to robust homeostasis/adaptation under inflow or outflow perturbations. Here we report that harmonic oscillations occur in models of such homeostatic controllers and that a close relationship exists between the control of the p53/Mdm2 system and that of a homeostatic inflow controller. This homeostatic control model of the p53 system provides an explanation why large fluctuations in the amplitude of p53/Mdm2 oscillations may arise as part of the homeostatic regulation of p53 by Mdm2 under DNA-damaging conditions. In the presence of DNA damage p53 is upregulated, but is subject to a tight control by Mdm2 and other factors to avoid a premature apoptotic response of the cell at low DNA damage levels. One of the regulatory steps is the Mdm2-mediated degradation of p53 by the proteasome. Oscillations in the p53/Mdm2 system are considered to be part of a mechanism by which a cell decides between cell cycle arrest/DNA repair and apoptosis. In the homeostatic inflow control model, harmonic oscillations in p53/Mdm2 levels arise when the binding strength of p53 to degradation complexes increases. Due to the harmonic character of the oscillations rapid fluctuating noise can lead, as experimentally observed, to large variations in the amplitude of the oscillation but not in their period, a behavior which has been difficult to simulate by deterministic limit-cycle models. In conclusion, the oscillatory response of homeostatic controllers may provide new insights into the origin and role of oscillations observed in homeostatically controlled molecular networks.

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Equilibrium sampling for biomolecules under mechanical tension.
Publication Date: 2010 Feb 17 PMID: 20159170
Authors: Zeng, X. - Hu, H. - Zhou, H. X. - Marszalek, P. E. - Yang, W.
Journal: Biophys J

In the studies of force-induced conformational transitions of biomolecules, the large timescale difference from experiments presents the challenge of obtaining convergent sampling for molecular dynamics simulations. To circumvent this fundamental problem, an approach combining the replica-exchange method and umbrella sampling (REM-US) was developed to simulate mechanical stretching of biomolecules under equilibrium conditions. Equilibrium properties of conformational transitions can be obtained directly from simulations without further assumptions. To test the performance, we carried out REM-US simulations of atomic force microscope (AFM) stretching and relaxing measurements on the polysaccharide pustulan, a (1-->6)-beta-D-glucan, which undergoes well-characterized rotameric transitions in the backbone bonds. With significantly enhanced sampling convergence and efficiency, the REM-US approach closely reproduced the equilibrium force-extension curves measured in AFM experiments. Consistent with the reversibility in the AFM measurements, the new approach generated identical force-extension curves in both stretching and relaxing simulations-an outcome not reported in previous studies, proving that equilibrium conditions were achieved in the simulations. REM-US may provide a robust approach to modeling of mechanical stretching on polysaccharides and even nucleic acids.

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Nanogap dielectric spectroscopy for aptamer-based protein detection.
Publication Date: 2010 Feb 17 PMID: 20159169
Authors: Mannoor, M. S. - James, T. - Ivanov, D. V. - Beadling, L. - Braunlin, W.
Journal: Biophys J

Among the various label-free methods for monitoring biomolecular interactions, capacitive sensors stand out due to their simple instrumentation and compatibility with multiplex formats. However, electrode polarization due to ion gradient formation and noise from solution conductance limited early dielectric spectroscopic measurements to high frequencies only, which in turn limited their sensitivity to biomolecular interactions, as the applied excitation signals were too fast for the charged macromolecules to respond. To minimize electrode polarization effects, capacitive sensors with 20 nm electrode separation were fabricated using silicon dioxide sacrificial layer techniques. The nanoscale separation of the capacitive electrodes in the sensor results in an enhanced overlapping of electrical double layers, and apparently a more ordered "ice-like" water structure. Such effects in turn reduce low frequency contributions from bulk sample resistance and from electrode polarization, and thus markedly enhance sensitivity toward biomolecular interactions. Using these nanogap capacitive sensors, highly sensitive, label-free aptamer-based detection of protein molecules is achieved.

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Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator.
Publication Date: 2010 Feb 17 PMID: 20159168
Authors: Herz, J. - Siffrin, V. - Hauser, A. E. - Brandt, A. U. - Leuenberger, T. - Radbruch, H. - Zipp, F. - Niesner, R. A.
Journal: Biophys J

Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.

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Susceptibility of different proteins to flow-induced conformational changes monitored with Raman spectroscopy.
Publication Date: 2010 Feb 17 PMID: 20159167
Authors: Ashton, L. - Dusting, J. - Imomoh, E. - Balabani, S. - Blanch, E. W.
Journal: Biophys J

By directly monitoring stirred protein solutions with Raman spectroscopy, the reversible unfolding of proteins caused by fluid shear is examined for several natural proteins with varying structural properties and molecular weight. While complete denaturation is not observed, a wide range of spectral variances occur for the different proteins, indicating subtle conformational changes that appear to be protein-specific. A number of significant overall trends are apparent from the study. For globular proteins, the overall extent of spectral variance increases with protein size and the proportion of beta-structure. For two less structured proteins, fetuin and alpha-casein, the observed changes are of relatively low magnitude, despite the greater molecular structural mobility of these proteins. This implies that other protein-specific factors, such as posttranslational modifications, may also be significant. Individual band changes occurring in the spectral profiles of each individual protein are also discussed in detail.

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Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments.
Publication Date: 2010 Feb 17 PMID: 20159166
Authors: Chung, H. S. - Louis, J. M. - Eaton, W. A.
Journal: Biophys J

Forster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity.

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Primary changes of the mechanical properties of Southern Bean Mosaic Virus upon calcium removal.
Publication Date: 2010 Feb 17 PMID: 20159165
Authors: Zink, M. - Grubmuller, H.
Journal: Biophys J

The mechanical properties of viral shells are crucial determinates for the pathway and mechanism by which the genetic material leaves the capsid during infection and have therefore been studied by atomic force microscopy as well as by atomistic simulations. The mechanical response to forces from inside the capsid are found to be relevant, especially after ion removal from the shell structure, which is generally assumed to be essential during viral infection; however, atomic force microscopy measurements are restricted to probing the capsids from outside, and the primary effect of ion removal is still inaccessible. To bridge this gap, we performed atomistic force-probe molecular dynamics simulations of the complete solvated icosahedral shell of Southern Bean Mosaic Virus and compared the distribution of elastic constants and yielding forces on the icosahedral shell for probing from inside with the distribution of outside mechanical properties obtained previously. Further, the primary effect of calcium removal on the mechanical properties on both sides, as well as on their spatial distribution, is quantified. Marked differences are seen particularly at the pentamer centers, although only small structural changes occur on the short timescales of the simulation. This unexpected primary effect, hence, precedes subsequent effects due to capsid swelling. In particular, assuming that genome release is preceded by an opening of capsomers instead of a complete capsid bursting, our observed weakening along the fivefold symmetry axes let us suggest pentamers as possible exit ports for RNA release.

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Nonlocal interactions are responsible for tertiary structure formation in staphylococcal nuclease.
Publication Date: 2010 Feb 17 PMID: 20159164
Authors: Kato, S. - Kamikubo, H. - Hirano, S. - Yamazaki, Y. - Kataoka, M.
Journal: Biophys J

Rapid molecular collapse mediated by nonlocal interactions is believed to be a crucial event for protein folding. To investigate the role of nonlocal interactions in tertiary structure formation, we performed a nonlocal interaction substitution mutation analysis on staphylococcal nuclease (SNase). Y54 and I139 of wild-type (WT) SNase and Delta140-149 were substituted by cysteine to form intramolecular disulfide bonds, respectively called WT-SS and Delta140-149-SS. Under physiological conditions, the reduced form of Delta140-149-SS appears to assume a denatured structure; in contrast, the oxidized form of Delta140-149-SS forms a native-like structure. From this result, we conclude that the C-terminal region participates in a nonlocal interaction that is indispensable for the native structure. Although the oxidized form of WT-SS assumes a more compact denatured structure under acidic conditions than the WT, the kinetic measurements reveal that the refolding reactions of both the reduced and oxidized forms of WT-SS are similar to those of the WT, suggesting that an intact nonlocal interaction is established within the dead time (22 ms). On the basis of these results, we propose that the native nonlocal contact established at the early stage of the folding process facilitates further secondary structure formation.

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Thermo- and mesostabilizing protein interactions identified by temperature-dependent statistical potentials.
Publication Date: 2010 Feb 17 PMID: 20159163
Authors: Folch, B. - Dehouck, Y. - Rooman, M.
Journal: Biophys J

The goal of controlling protein thermostability is tackled here through establishing, by in silico analyses, the relative weight of residue-residue interactions in proteins as a function of temperature. We have designed for that purpose a (melting-) temperature-dependent, statistical distance potential, where the interresidue distances are computed between the side-chain geometric centers or their functional centers. Their separate derivation from proteins of either high or low thermal resistance reveals the interactions that contribute most to stability in different temperature ranges. Thermostabilizing interactions include salt bridges and cation-pi interactions (especially those involving arginine), aromatic interactions, and H-bonds between negatively charged and some aromatic residues. In contrast, H-bonds between two polar noncharged residues or between a polar noncharged residue and a negatively charged residue are relatively less stabilizing at high temperatures. An important observation is that it is necessary to consider both repulsive and attractive interactions in overall thermostabilization, as the degree of repulsion may also vary with temperature. These temperature-dependent potentials are not only useful for the identification of meso- and thermostabilizing pair interactions, but also exhibit predictive power, as illustrated by their ability to predict the melting temperature of a protein based on the melting temperature of homologous proteins.

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Exploring the contribution of collective motions to the dynamics of forced-unfolding in tubulin.
Publication Date: 2010 Feb 17 PMID: 20159162
Authors: Joshi, H. - Momin, F. - Haines, K. E. - Dima, R. I.
Journal: Biophys J

Decomposition of the intrinsic dynamics of proteins into collective motions among distant regions of the protein structure provides a physically appealing approach that couples the dynamics of the system with its functional role. The cellular functions of microtubules (an essential component of the cytoskeleton in all eukaryotic cells) depend on their dynamic instability, which is altered by various factors among which applied forces are central. To shed light on the coupling between forces and the dynamic instability of microtubules, we focus on the investigation of the response of the microtubule subunits (tubulin) to applied forces. We address this point by adapting an approach designed to survey correlations for the equilibrium dynamics of proteins to the case of correlations for proteins forced-dynamics. The resulting collective motions in tubulin have a number of functional implications, such as the identification of long-range couplings with a role in blocking the dynamic instability of microtubules. A fundamental implication of our study for the life of a cell is that, to increase the likelihood of unraveling of large cytoskeletal filaments under physiological forces, molecular motors must use a combination of pulling and torsion rather than just pulling.

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(Un)Folding mechanisms of the FBP28 WW domain in explicit solvent revealed by multiple rare event simulation methods.
Publication Date: 2010 Feb 17 PMID: 20159161
Authors: Juraszek, J. - Bolhuis, P. G.
Journal: Biophys J

We report a numerical study of the (un)folding routes of the truncated FBP28 WW domain at ambient conditions using a combination of four advanced rare event molecular simulation techniques. We explore the free energy landscape of the native state, the unfolded state, and possible intermediates, with replica exchange molecular dynamics. Subsequent application of bias-exchange metadynamics yields three tentative unfolding pathways at room temperature. Using these paths to initiate a transition path sampling simulation reveals the existence of two major folding routes, differing in the formation order of the two main hairpins, and in hydrophobic side-chain interactions. Having established that the hairpin strand separation distances can act as reasonable reaction coordinates, we employ metadynamics to compute the unfolding barriers and find that the barrier with the lowest free energy corresponds with the most likely pathway found by transition path sampling. The unfolding barrier at 300 K is approximately 17 k(B)T approximately 42 kJ/mol, in agreement with the experimental unfolding rate constant. This work shows that combining several powerful simulation techniques provides a more complete understanding of the kinetic mechanism of protein folding.

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TEM-1 backbone dynamics-insights from combined molecular dynamics and nuclear magnetic resonance.
Publication Date: 2010 Feb 17 PMID: 20159160
Authors: Fisette, O. - Morin, S. - Savard, P. Y. - Lague, P. - Gagne, S. M.
Journal: Biophys J

Dynamic properties of class A beta-lactamase TEM-1 are investigated from molecular dynamics (MD) simulations. Comparison of MD-derived order parameters with those obtained from model-free analysis of nuclear magnetic resonance (NMR) relaxation data shows high agreement for N-H moieties within alpha- and beta-secondary structures, but significant deviation for those in loops. This was expected, because motions slower than the protein global tumbling often take place in loop regions. As previously shown using NMR, TEM-1 is a highly ordered protein. Motions are observed within the Omega loop that could, upon substrate binding, stabilize E166 in a catalytically efficient position as the cavity between the protein core and the Omega loop is partially filled. The rigidity of active site residues is consistent with the enzyme high turnover number. MD data are also shown to be useful during the model selection step of model-free analysis: local N-H motions observed over the course of the trajectories help assess whether a peptide plan undergoes low or high amplitude motions on one or more timescales. This joint use of MD and NMR provides a better description of protein dynamics than would be possible using either technique alone.

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Free energy profile of RNA hairpins: a molecular dynamics simulation study.
Publication Date: 2010 Feb 17 PMID: 20159159
Authors: Deng, N. J. - Cieplak, P.
Journal: Biophys J

RNA hairpin loops are one of the most abundant secondary structure elements and participate in RNA folding and protein-RNA recognition. To characterize the free energy surface of RNA hairpin folding at an atomic level, we calculated the potential of mean force (PMF) as a function of the end-to-end distance, by using umbrella sampling simulations in explicit solvent. Two RNA hairpins containing tetraloop cUUCGg and cUUUUg are studied with AMBER ff99 and CHARMM27 force fields. Experimentally, the UUCG hairpin is known to be significantly more stable than UUUU. In this study, the calculations using AMBER force field give a qualitatively correct description for the folding of two RNA hairpins, as the calculated PMF confirms the global stability of the folded structures and the resulting relative folding free energy is in quantitative agreement with the experimental result. The hairpin stabilities are also correctly differentiated by the more rapid molecular mechanics-Poisson Boltzmann-surface area approach, but the relative free energy estimated from this method is overestimated. The free energy profile shows that the native state basin and the unfolded state plateau are separated by a wide shoulder region, which samples a variety of native-like structures with frayed terminal basepair. The calculated PMF lacks major barriers that are expected near the transition regions, and this is attributed to the limitation of the 1-D reaction coordinate. The PMF results are compared with other studies of small RNA hairpins using kinetics method and coarse grained models. The two RNA hairpins described by CHARMM27 are significantly more deformable than those represented by AMBER. Compared with the AMBER results, the CHARMM27 calculated DeltaG(fold) for the UUUU tetraloop is in better agreement with the experimental results. However, the CHARMM27 calculation does not confirm the global stability of the experimental UUCG structure; instead, the extended conformations are predicted to be thermodynamically stable in solution. This finding is further supported by separate unrestrained CHARMM27 simulations, in which the UUCG hairpin unfolds spontaneously within 10 ns. The instability of the UUCG hairpin originates from the loop region, and propagates to the stem. The results of this study provide a molecular picture of RNA hairpin unfolding and reveal problems in the force field descriptions for the conformational energy of certain RNA hairpin.

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Material properties of Caenorhabditis elegans swimming at low Reynolds number.
Publication Date: 2010 Feb 17 PMID: 20159158
Authors: Sznitman, J. - Purohit, P. K. - Krajacic, P. - Lamitina, T. - Arratia, P. E.
Journal: Biophys J

Undulatory locomotion, as seen in the nematode Caenorhabditis elegans, is a common swimming gait of organisms in the low Reynolds number regime, where viscous forces are dominant. Although the nematode's motility is expected to be a strong function of its material properties, measurements remain scarce. Here, the swimming behavior of C. elegans is investigated in experiments and in a simple model. Experiments reveal that nematodes swim in a periodic fashion and generate traveling waves that decay from head to tail. The model is able to capture the experiments' main features and is used to estimate the nematode's Young's modulus E and tissue viscosity eta. For wild-type C. elegans, we find E approximately 3.77 kPa and eta approximately -860 Pa.s; values of eta for live C. elegans are negative because the tissue is generating rather than dissipating energy. Results show that material properties are sensitive to changes in muscle functional properties, and are useful quantitative tools with which to more accurately describe new and existing muscle mutants.

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Microarchitecture is severely compromised but motor protein function is preserved in dystrophic mdx skeletal muscle.
Publication Date: 2010 Feb 17 PMID: 20159157
Authors: Friedrich, O. - Both, M. - Weber, C. - Schurmann, S. - Teichmann, M. D. - von Wegner, F. - Fink, R. H. - Vogel, M. - Chamberlain, J. S. - Garbe, C.
Journal: Biophys J

Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.

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Quantifying biomolecule diffusivity using an optimal bayesian method.
Publication Date: 2010 Feb 17 PMID: 20159156
Authors: Voisinne, G. - Alexandrou, A. - Masson, J. B.
Journal: Biophys J

We propose a Bayesian method to extract the diffusivity of biomolecules evolving freely or inside membrane microdomains. This approach assumes a model of motion for the particle considered, namely free Brownian motion or confined diffusion. In each framework, a systematic Bayesian scheme is provided for estimating the diffusivity. We show that this method reaches the best performances theoretically achievable. Its efficiency overcomes that of widely used methods based on the analysis of the mean-square displacement. The approach presented here also gives direct access to the uncertainty on the estimation of the diffusivity and predicts the number of steps of the trajectory necessary to achieve any desired precision. Its robustness with respect to noise on the position of the biomolecule is also investigated.

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Partitioning of nonsteroidal antiinflammatory drugs in lipid membranes: a molecular dynamics simulation study.
Publication Date: 2010 Feb 17 PMID: 20159155
Authors: Boggara, M. B. - Krishnamoorti, R.
Journal: Biophys J

Using the potential of mean constrained force method, molecular dynamics simulations with atomistic details were performed to examine the partitioning and nature of interactions of two nonsteroidal antiinflammatory drugs, namely aspirin and ibuprofen, in bilayers of dipalmitoylphosphatidylcholine. Two charge states (neutral and anionic) of the drugs were simulated to understand the effect of protonation or pH on drug partitioning. Both drugs, irrespective of their charge state, were found to have high partition coefficients in the lipid bilayer from water. However, the values and trends of the free energy change and the location of the minima in the bilayer are seen to be influenced by the drug structure and charge state. In the context of the transport of the drugs through the bilayer, the charged forms were found to permeate fully hydrated in contrast to the neutral forms that permeate unhydrated.

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Pardaxin permeabilizes vesicles more efficiently by pore formation than by disruption.
Publication Date: 2010 Feb 17 PMID: 20159154
Authors: Vad, B. S. - Bertelsen, K. - Johansen, C. H. - Pedersen, J. M. - Skrydstrup, T. - Nielsen, N. C. - Otzen, D. E.
Journal: Biophys J

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance (13)C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.

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Bridging timescales and length scales: from macroscopic flux to the molecular mechanism of antibiotic diffusion through porins.
Publication Date: 2010 Feb 17 PMID: 20159153
Authors: Hajjar, E. - Mahendran, K. R. - Kumar, A. - Bessonov, A. - Petrescu, M. - Weingart, H. - Ruggerone, P. - Winterhalter, M. - Ceccarelli, M.
Journal: Biophys J

Our aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for beta-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway. We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation. The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates.

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The molecular determinants of the increased reduction potential of the rubredoxin domain of rubrerythrin relative to rubredoxin.
Publication Date: 2010 Feb 17 PMID: 20159152
Authors: Luo, Y. - Ergenekan, C. E. - Fischer, J. T. - Tan, M. L. - Ichiye, T.
Journal: Biophys J

Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the rubredoxin domain of rubrerythrin over rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of rubredoxin and rubrerythrin and molecular dynamics simulations of rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn(160) and His(179) in rubrerythrin versus valines in rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala(176) in rubrerythrin versus a leucine in rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer.

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Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli.
Publication Date: 2010 Feb 17 PMID: 20159151
Authors: Kumar, M. - Mommer, M. S. - Sourjik, V.
Journal: Biophys J

Protein mobility affects most cellular processes, such as the rates of enzymatic reactions, signal transduction, and assembly of macromolecular complexes. Despite such importance, little systematic information is available about protein diffusion inside bacterial cells. Here we combined fluorescence recovery after photobleaching with numerical modeling to analyze mobility of a set of fluorescent protein fusions in the bacterial cytoplasm, the plasma membrane, and in the nucleoid. Estimated diffusion coefficients of cytoplasmic and membrane proteins show steep dependence on the size and on the number of transmembrane helices, respectively. Protein diffusion in both compartments is thus apparently obstructed by a network of obstacles, creating the so-called molecular sieving effect. These obstructing networks themselves, however, appear to be dynamic and allow a slow and nearly size-independent movement of large proteins and complexes. The obtained dependencies of protein mobility on the molecular mass and the number of transmembrane helices can be used as a reference to predict diffusion rates of proteins in Escherichia coli. Mobility of DNA-binding proteins apparently mainly depends on their binding specificity, with FRAP recovery kinetics being slower for the highly specific TetR repressor than for the relatively nonspecific H-NS regulator.

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Cell adhesion strength is controlled by intermolecular spacing of adhesion receptors.
Publication Date: 2010 Feb 17 PMID: 20159150
Authors: Selhuber-Unkel, C. - Erdmann, T. - Lopez-Garcia, M. - Kessler, H. - Schwarz, U. S. - Spatz, J. P.
Journal: Biophys J

Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings > or = 90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings < or = 50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength.

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Strength dependence of cadherin-mediated adhesions.
Publication Date: 2010 Feb 17 PMID: 20159149
Authors: Ladoux, B. - Anon, E. - Lambert, M. - Rabodzey, A. - Hersen, P. - Buguin, A. - Silberzan, P. - Mege, R. M.
Journal: Biophys J

Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands. On the most rigid substrates, cells appear well-spread and present cadherin adhesions and cytoskeletal organization similar to those classically observed on cadherin-coated glass substrates. However, when cells are cultured on softer substrates, a change in morphology is observed: the cells are less spread, with a more disorganized actin network. A quantitative analysis of the cells adhering on the cadherin-coated surfaces shows that forces are correlated with the formation of cadherin adhesions. The stiffer the substrates, the larger are the average traction forces and the more developed are the cadherin adhesions. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechanosensitive regulation of cadherin-mediated intercellular junctions through the cellular contractile machinery.

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An analytic solution of the cable equation predicts frequency preference of a passive shunt-end cylindrical cable in response to extracellular oscillating electric fields.
Publication Date: 2010 Feb 17 PMID: 20159148
Authors: Monai, H. - Omori, T. - Okada, M. - Inoue, M. - Miyakawa, H. - Aonishi, T.
Journal: Biophys J

Under physiological and artificial conditions, the dendrites of neurons can be exposed to electric fields. Recent experimental studies suggested that the membrane resistivity of the distal apical dendrites of cortical and hippocampal pyramidal neurons may be significantly lower than that of the proximal dendrites and the soma. To understand the behavior of dendrites in time-varying extracellular electric fields, we analytically solved cable equations for finite cylindrical cables with and without a leak conductance attached to one end by employing the Green's function method. The solution for a cable with a leak at one end for direct-current step electric fields shows a reversal in polarization at the leaky end, as has been previously shown by employing the separation of variables method and Fourier series expansion. The solution for a cable with a leak at one end for alternating-current electric fields reveals that the leaky end shows frequency preference in the response amplitude. Our results predict that a passive dendrite with low resistivity at the distal end would show frequency preference in response to sinusoidal extracellular local field potentials. The Green's function obtained in our study can be used to calculate response for any extracellular electric field.

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Drag force as a tool to test the active mechanical response of PC12 neurites.
Publication Date: 2010 Feb 17 PMID: 20159147
Authors: Bernal, R. - Melo, F. - Pullarkat, P. A.
Journal: Biophys J

We investigate the mechanical response of PC12 neurites subjected to a drag force imposed by a laminar flow perpendicular to the neurite axis. The curvature of the catenary shape acquired by an initially straight neurite under the action of the drag force provides information on both elongation and tension of the neurite. This method allows us to measure the rest tension and viscoelastic parameters of PC12 neurites and active behavior of neurites. Measurement of oscillations in the strain rate of neurites at constant flow rate provides insight on the response of molecular motors and additional support for the presence of a negative strain-rate sensitivity region in the global mechanical response of PC12 neurites.

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Immune response modeling of interferon beta-pretreated influenza virus-infected human dendritic cells.
Publication Date: 2010 Feb 17 PMID: 20159146
Authors: Qiao, L. - Phipps-Yonas, H. - Hartmann, B. - Moran, T. M. - Sealfon, S. C. - Hayot, F.
Journal: Biophys J

The pretreatment of human dendritic cells with interferon-beta enhances their immune response to influenza virus infection. We measured the expression levels of several key players in that response over a period of 13 h both during pretreatment and after viral infection. Their activation profiles reflect the presence of both negative and positive feedback loops in interferon induction and interferon signaling pathway. Based on these measurements, we have developed a comprehensive computational model of cellular immune response that elucidates its mechanism and its dynamics in interferon-pretreated dendritic cells, and provides insights into the effects of duration and strength of pretreatment.

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A phase function to quantify serial dependence between discrete samples.
Publication Date: 2010 Feb 17 PMID: 20159145
Authors: Dodla, R. - Wilson, C. J.
Journal: Biophys J

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