Biophysical Journal current issue

[BIOPHYSICAL LETTERS] Ten-Microsecond Molecular Dynamics Simulation of a Fast-Folding WW Domain

Freddolino, P. L., Liu, F., Gruebele, M., Schulten, K. — 2008-04-23

All-atom molecular dynamics (MD) simulations of protein folding allow analysis of the folding process at an unprecedented level of detail. Unfortunately, such simulations have not yet reached their full potential both due to difficulties in sufficiently sampling the microsecond timescales needed for folding, and because the force field used may yield neither the correct dynamical sequence of events nor the folded structure. The ongoing study of protein folding through computational methods thus requires both improvements in the performance of molecular dynamics programs to make longer timescales accessible, and testing of force fields in the context of folding simulations. We report a ten-microsecond simulation of an incipient downhill-folding WW domain mutant along with measurement of a molecular time and activated folding time of 1.5 microseconds and 13.3 microseconds, respectively. The protein simulated in explicit solvent exhibits several metastable states with incorrect topology and does not assume the native state during the present simulations.

[BIOPHYSICAL LETTERS] Nonlinear Relationship between Level of Blood Flow and Skin Temperature for Different Dynamics of Temperature Change

Vuksanovic, V., Sheppard, L. W., Stefanovska, A. — 2008-04-23

We present a study of the relationship between blood flow and skin temperature under different dynamics of skin-temperature-change: locally induced thermal shock and well controlled, gradual change. First, we demonstrate memory phenomena for blood flow and skin temperature under both conditions. Secondly, we point out that the "hysteresis" loops obtained are dependent on initial conditions, indicating physiological response times of more than twenty minutes. We also show that under thermal shock the level of blood flow is preserved up to some characteristic temperature limit, independently of subject.

[NEW AND NOTABLE] "Shimmying Like My Sister" Cell

Curtis, A. — 2008-04-23

[BIOPHYSICAL THEORY AND MODELING] Fast In Silico Protein Folding by Introduction of Alternating Hydrogen Bond Potentials

Wolf, M. G., de Leeuw, S. W. — 2008-04-23

We accelerate protein folding in all-atom molecular dynamics simulations by introducing alternating hydrogen bond potentials as a supplement to the force field. The alternating hydrogen bond potentials result in accelerated hydrogen bond reordering, which leads to rapid formation of secondary structure elements. The method does not require knowledge of the native state but generates the potentials based on the development of the tertiary structure in the simulation. In protein folding, the formation of secondary structure elements, especially -helix and β-sheet, is very important, and we show that our method can fold both efficiently and with great speed.

[BIOPHYSICAL THEORY AND MODELING] Coarse-Grained Molecular Simulation of Diffusion and Reaction Kinetics in a Crowded Virtual Cytoplasm

2008-04-23

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.

[BIOPHYSICAL THEORY AND MODELING] Colloid Adsorption onto Responsive Membranes

Dias, R. S., Linse, P. — 2008-04-23

The adsorption of colloids of varying sizes and charges onto a surface that carries both negative and positive charges, representing a membrane, has been investigated using a simple model employing Monte Carlo simulations. The membrane is made of positive and negative charges (headgroups) that are allowed to move along the membrane, simulating the translational diffusion of the lipids, and are also allowed to protrude into the solution, giving rise to a fluid and soft membrane. When an uncharged colloid is placed in the vicinity of the membrane, a short-range repulsion between the colloid and the membrane is observed and the membrane will deflect to avoid coming into contact with the colloid. When the colloid is charged, the membrane response is twofold: the headgroups of the membrane move toward the colloid, as if to partly embrace it, and the positive headgroups of the membrane approach the oppositely charged colloid, inducing the demixing of the membrane lipids (polarization). The presence of protrusions enhances the polarization of the membrane. Potential of mean force calculations show that protrusions give rise to a more long-range attractive colloid-membrane potential which has a smaller magnitude at short separations.

[BIOPHYSICAL THEORY AND MODELING] The Intrinsic Dynamics and Function of Nickel-Binding Regulatory Protein: Insights from Elastic Network Analysis

Cui, G., Merz, K. M. — 2008-04-23

Nickel-responsive protein NikR regulates the nickel uptake in nickel-dependent bacteria by interacting with the operator of nikABCDE and subsequently repressing the transcription of NikABCDE, an ABC-type nickel transporter system. The function of NikR and its affinity for the operator DNA is highly conformation-dependent, which has been confirmed by three independent crystallographic studies on NikR proteins from different bacteria. Depending on the intracellular nickel concentration, NikR is able to adopt either the open form or one of the two closed forms (cis and trans) that differ in the domain-domain arrangement. Only the closed cis form is optimal for DNA binding. We examined the low-resolution vibrational spectrum of NikR in each conformational form using the elastic network model and observed large-scale domain-domain vibrations that are closely related to the conformational transitions required for function, particularly the symmetric bending mode and the asymmetric twisting mode. This analysis on the intrinsic dynamics coded in the three-dimensional molecular construct allows us to examine the proposed mechanisms of NikR regulation from the standpoint of protein collective motions. Our findings further support the three-state equilibrium hypothesis proposed by others, and imply that an isolated closed cis form may be dynamically unstable but can be stabilized by DNA binding. However, we also found that the simple C-model used in the current analysis is insufficient to capture the impact of nickel binding on the protein dynamics, for which an all-atom model with detailed atom typing is more appropriate.

[BIOPHYSICAL THEORY AND MODELING] The Elastic Properties of the Structurally Characterized Myosin II S2 Subdomain: A Molecular Dynamics and Normal Mode Analysis

Adamovic, I., Mijailovich, S. M., Karplus, M. — 2008-04-23

The elastic properties (stretching and bending moduli) of myosin are expected to play an important role in its function. Of particular interest is the extended -helical coiled-coil portion of the molecule. Since there is no high resolution structure for the entire coiled-coil, a study is made of the scallop myosin II S2 subdomain for which an x-ray structure is available (Protein Data Bank 1nkn). We estimate the stretching and bending moduli of the S2 subdomain with an atomic level model by use of molecular simulations. Results were obtained from nonequilibrium molecular dynamics simulations in the presence of an external force, from the fluctuations in equilibrium molecular dynamics simulations and from normal modes. In addition, a poly-Ala (78 amino acid residues) -helix model was examined to test the methodology and because of its interest as part of the lever arm. As expected, both the -helix and coiled-coil S2 subdomain are very stiff for stretching along the main axis, with the stretching stiffness constant in the range 60–80 pN/nm (scaled to the 60 nm long S2). Both molecules are much more flexible for bending with a lateral stiffness of ~0.010pN/nm for the S2 and 0.0055pN/nm for the -helix (scaled to 60 nm). These results are expected to be useful in estimating cross-bridge elasticity, which is required for understanding the strain-dependent transitions in the actomyosin cycle and for the development of three-dimensional models of muscle contraction.

[BIOPHYSICAL THEORY AND MODELING] The Receptor-Mediated Endocytosis of Nonspherical Particles

Decuzzi, P., Ferrari, M. — 2008-04-23

Enveloped viruses and nanosized biomimetic particles for drug and gene delivery enter target cells mainly through receptor-mediated endocytosis. A few models have been presented to elucidate the mechanics of particle engulfment by the cell membrane, showing how size and surface chemico-physical properties favor or oppose internalization. In this work, the effect of particle nonsphericity is addressed considering elliptical cylindrical particles with aspect ratio . Using a continuum energetic approach, three different conditions have been identified: for sufficiently small , the particle is not even wrapped by the cell membrane; for sufficiently large , the particle is partially wrapped ("frustrated endocytosis"); and for intermediate values of , the particle is fully wrapped and eventually internalized. Given the pleomorphism of viruses and the broad spectrum of shapes for nanosized biomimetic particles, the results presented may be of interest to virologists, pharmacologists, toxicologists, and nanotechnologists.

[BIOPHYSICAL THEORY AND MODELING] Simulation of Nitroxide Electron Paramagnetic Resonance Spectra from Brownian Trajectories and Molecular Dynamics Simulations

DeSensi, S. C., Rangel, D. P., Beth, A. H., Lybrand, T. P., Hustedt, E. J. — 2008-04-23

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.

[BIOPHYSICAL THEORY AND MODELING] Depolymerization-Driven Flow in Nematode Spermatozoa Relates Crawling Speed to Size and Shape

Zajac, M., Dacanay, B., Mohler, W. A., Wolgemuth, C. W. — 2008-04-23

Cell crawling is an inherently physical process that includes protrusion of the leading edge, adhesion to the substrate, and advance of the trailing cell body. Research into advance of the cell body has focused on actomyosin contraction, with cytoskeletal disassembly regarded as incidental, rather than causative; however, extracts from nematode spermatozoa, which use Major Sperm Protein rather than actin, provide at least one example where cytoskeletal disassembly apparently generates force in the absence of molecular motors. To test whether depolymerization can explain force production during nematode sperm crawling, we constructed a mathematical model that simultaneously describes the dynamics of both the cytoskeleton and the cytosol. We also performed corresponding experiments using motile Caenorhabditis elegans spermatozoa. Our experiments reveal that crawling speed is an increasing function of both cell size and anterior-posterior elongation. The quantitative, depolymerization-driven model robustly predicts that cell speed should increase with cell size and yields a cytoskeletal disassembly rate that is consistent with previous measurements. Notably, the model requires anisotropic elasticity, with the cell being stiffer along the direction of motion, to accurately reproduce the dependence of speed on elongation. Our simulations also predict that speed should increase with cytoskeletal anisotropy and disassembly rate.

[BIOPHYSICAL THEORY AND MODELING] Molecular Dynamics Simulations of the Photoactive Protein Nitrile Hydratase

Kubiak, K., Nowak, W. — 2008-04-23

Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, and β. Hydrogen bonds between βArg-56 and Cys-114 sulfenic acid (CEA114) are important to maintain the enzymatic activity. The enzyme may be inactivated by endogenous nitric oxide (NO) and activated by absorption of photons of wavelength < 630 nm. To explain the photosensitivity and to propose structural determinants of catalytic activity, differences in the dynamics of light-active and dark-inactive forms of NHase were investigated using molecular dynamics (MD) modeling. To this end, a new set of force field parameters for nonstandard NHase active sites have been developed. The dynamics of the photodissociated NO ligand in the enzyme channel was analyzed using the locally enhanced sampling method, as implemented in the MOIL MD package. A series of 1 ns trajectories of NHases shows that the protonation state of the active site affects the dynamics of the catalytic water and NO ligand close to the metal center. MD simulations support the catalytic mechanism in which a water molecule bound to the metal ion directly attacks the nitrile carbon.

[BIOPHYSICAL THEORY AND MODELING] The Stochastic Dynamics of Filopodial Growth

Lan, Y., Papoian, G. A. — 2008-04-23

A filopodium is a cytoplasmic projection, exquisitely built and regulated, which extends from the leading edge of the migrating cell, exploring the cell's neighborhood. Commonly, filopodia grow and retract after their initiation, exhibiting rich dynamical behaviors. We model the growth of a filopodium based on a stochastic description which incorporates mechanical, physical, and biochemical components. Our model provides a full stochastic treatment of the actin monomer diffusion and polymerization of each individual actin filament under stress of the fluctuating membrane. We investigated the length distribution of individual filaments in a growing filopodium and studied how it depends on various physical parameters. The distribution of filament lengths turned out to be narrow, which we explained by the negative feedback created by the membrane load and monomeric G-actin gradient. We also discovered that filopodial growth is strongly diminished upon increasing retrograde flow, suggesting that regulating the retrograde flow rate would be a highly efficient way to control filopodial extension dynamics. The filopodial length increases as the membrane fluctuations decrease, which we attributed to the unequal loading of the membrane force among individual filaments, which, in turn, results in larger average polymerization rates. We also observed significant diffusional noise of G-actin monomers, which leads to smaller G-actin flux along the filopodial tube compared with the prediction using the diffusion equation. Overall, partial cancellation of these two fluctuation effects allows a simple mean field model to rationalize most of our simulation results. However, fast fluctuations significantly renormalize the mean field model parameters. The biological significance of our filopodial model and avenues for future development are also discussed.

[BIOPHYSICAL THEORY AND MODELING] A Unification of the Elastic Network Model and the Gaussian Network Model for Optimal Description of Protein Conformational Motions and Fluctuations

Zheng, W. — 2008-04-23

Coarse-grained elastic models with a C-only representation and harmonic interactions have been increasingly used to describe the conformational motions and flexibility of various proteins. In this work, we will unify two complementary elastic models—the elastic network model (ENM) and the Gaussian network model (GNM), in the framework of a generalized anisotropic network model (G-ANM) with a new anisotropy parameter, f_anm. The G-ANM is reduced to GNM at f_anm = 1, and ENM at f_anm = 0. By analyzing a list of protein crystal structure pairs using G-ANM, we have attained optimal descriptions of both the isotropic thermal fluctuations and the crystallographically observed conformational changes with a small f_anm (f_anm ≤ 0.1) and a physically realistic cutoff distance, R_c ~ 8 Å. Thus, the G-ANM improves the performance of GNM and ENM while preserving their simplicity. The properly parameterized G-ANM will enable more accurate and realistic modeling of protein conformational motions and flexibility.

[BIOPHYSICAL THEORY AND MODELING] Thermal Unfolding Simulations of Bacterial Flagellin: Insight into its Refolding Before Assembly

Chng, C.-P., Kitao, A. — 2008-04-23

Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded β-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.

[BIOPHYSICAL THEORY AND MODELING] Molecular Models Predict Light-Induced Glutamine Tautomerization in BLUF Photoreceptors

Domratcheva, T., Grigorenko, B. L., Schlichting, I., Nemukhin, A. V. — 2008-04-23

The recently discovered photoreceptor proteins containing BLUF (sensor of blue light using FAD) domains mediate physiological responses to blue light in bacteria and euglena. In BLUF domains, blue light activates the flavin chromophore yielding a signaling state characterized by a ~10 nm red-shifted absorption. We developed molecular models for the dark and light states of the BLUF domain of the Rhodobacter sphaeroides AppA protein, which are based on the crystal structures and quantum-mechanical simulations. According to these models, photon absorption by the flavin results in a tautomerization and 180° rotation of the Gln side chain that interacts with the flavin cofactor. This chemical modification of the Gln residue induces alterations in the hydrogen bond network in the core of the photoreceptor domain, which were observed in numerous spectroscopic experiments. The calculated electronic transition energies and vibrational frequencies of the proposed dark and light states are consistent with the optical and IR spectral changes observed during the photocycle. Light-induced isomerization of an amino acid residue instead of a chromophore represents a feature that has not been described previously in photoreceptors.

[BIOPHYSICAL THEORY AND MODELING] Nanomechanical Model of Microtubule Translocation in the Presence of Electric Fields

Kim, T., Kao, M.-T., Hasselbrink, E. F., Meyhofer, E. — 2008-04-23

Research efforts in recent years have been directed toward actively controlling the direction of translocation of microtubules on a kinesin-coated glass surface with E-fields (electric fields), opening up the possibility of engineering controllable nanodevices that integrate microtubules and motor proteins into their function. Here, we present a detailed, biophysical model that quantitatively describes our observations on the steering of microtubules by electric fields. A sudden application of an electric field parallel to the surface and normal to the translocation direction of a microtubule bends the leading end toward the anode, because Coulombic (electrophoretic) forces are dominant on negatively charged microtubules. Modeling this bending as a cantilever deflection with uniform loading requires accurate mechanical and electrical properties of microtubules, including their charge density, viscous drag, and flexural rigidity. We determined the charge density of microtubules from measurements of the electrophoretic mobility in a "zero flow" capillary electrophoresis column and estimate it to be 256 e^– per micron of length. Viscous drag forces on deflecting microtubules in electroosmotic flows were studied theoretically and experimentally by directly characterizing flows using a caged dye imaging method. The flexural rigidity of microtubules was measured by applying E-fields to microtubules with biotinylated segments that were bound to streptavidin-coated surfaces. From the calculated loading, and the Bernoulli-Euler curvature and moment equation, we find that the flexural rigidity of microtubules depends on their length, suggesting microtubules are anisotropic. Finally, our model accurately predicts the biophysical properties and behavior of microtubules directed by E-fields, which opens new avenues for the design of biomolecular nanotransport systems.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Thermodynamic and Kinetic Properties of Amino-Terminal and S4-S5 Loop HERG Channel Mutants under Steady-State Conditions

Alonso-Ron, C., de la Pena, P., Miranda, P., Dominguez, P., Barros, F. — 2008-04-23

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K⁺ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z_g values and a less negative G_o, indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased G_o, indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Osmotic Water Transport with Glucose in GLUT2 and SGLT

Naftalin, R. J. — 2008-04-23

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.

[MEMBRANES] Surface Rheology and Phase Transitions of Monolayers of Phospholipid/Cholesterol Mixtures

Vranceanu, M., Winkler, K., Nirschl, H., Leneweit, G. — 2008-04-23

The dynamic surface elasticity and the surface dilational viscosity of three binary phospholipid/cholesterol mixtures were determined with axisymmetric drop shape analysis on a harmonically oscillating pendent drop. Dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine were used to explore the rheological properties and phase transitions of mixtures of saturated and unsaturated phospholipids with cholesterol. The growth rates for surface dilational viscosity and dynamic elasticity are parallel for all film pressures studied. Characteristic breaks and plateaus could be found for these growth rates, indicating phase transitions. For dipalmitoylphosphatidylcholine/cholesterol and dimyristoylphosphatidylcholine/cholesterol mixtures, phase diagrams with six regions separated by phase boundaries were found, which are in good agreement with phase transitions reported in the literature for static measurements of isotherms and isobars on a Langmuir film balance and from fluorescence microscopy. Some phase boundaries were only found by dynamic, but not by static, elasticity measurements. Imaging methods revealed phase separations produced by the formation of condensed stoichiometric complexes leading to micron-sized and mostly circular domains. The effects of these complexes on monolayer rheology in liquid/liquid phases is described. Furthermore, liquid/solid and solid phase transitions are discussed.

[MEMBRANES] Differential Modulation of Membrane Structure and Fluctuations by Plant Sterols and Cholesterol

Hodzic, A., Rappolt, M., Amenitsch, H., Laggner, P., Pabst, G. — 2008-04-23

We have studied the concentration and temperature dependent influence of cholesterol, stigmasterol, and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl phosphatidylcholine and palmitoyl oleoyl phosphatidylcholine bilayers applying small-angle x-ray scattering, as well as dilatometry and ultrasound velocimetry. Independent of the lipid matrix, cholesterol was found to be most efficient in modulating bilayer thickness and elasticity, followed by sitosterol and stigmasterol. This can be attributed to the additional ethyl groups and double bond at the C₁₇ alkyl side-chain of the two plant sterols. Hence, it seems that some flexibility of the sterol hydrocarbon chain is needed to accommodate within the lipid bilayer. In addition, we did not observe two populations of membranes within the putative liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. Instead, the diffraction patterns could be interpreted in terms of a uniform phase. This lends further support to the idea of compositional fluctuations of unstable sterol rich domains recently brought up by fluorescence microscopy experiments, which contrasts the formation of stable domains within the miscibility gap of binary lipid/sterol mixtures.

[MEMBRANES] Site-Directed Fluorescence Labeling of a Membrane Protein with BADAN: Probing Protein Topology and Local Environment

Koehorst, R. B. M., Spruijt, R. B., Hemminga, M. A. — 2008-04-23

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly -helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.

[MEMBRANES] Probing Receptor-Translocator Interactions in the Oligopeptide ABC Transporter by Fluorescence Correlation Spectroscopy

Doeven, M. K., van den Bogaart, G., Krasnikov, V., Poolman, B. — 2008-04-23

The oligopeptide transporter Opp is a five-component ABC uptake system. The extracytoplasmic lipid-anchored substrate-binding protein (or receptor) OppA delivers peptides to an integral membrane complex OppBCDF (or translocator), where, on ATP binding and hydrolysis, translocation across the membrane takes place. OppA and OppBCDF were labeled with fluorescent probes, reconstituted into giant unilamellar vesicles, and the receptor-translocator interactions were investigated by fluorescence correlation spectroscopy. Lateral mobility of OppA was reduced on incorporation of OppBCDF into giant unilamellar vesicles, and decreased even further on the addition of peptide. Fluorescence cross-correlation measurements revealed that OppBCDF distinguished liganded from unliganded OppA, binding only the former. Addition of ATP or its nonhydrolyzable analog AMP-PNP resulted in release of OppA from OppBCDF. In vanadate-trapped "transition state" conditions, OppA also was not bound by OppBCDF. A model is presented in which ATP-binding to OppDF results in donation of peptide to OppBC and simultaneous release of OppA. ATP-hydrolysis would complete the peptide translocation and reset the transporter for another catalytic cycle. Implications in terms of a general transport mechanism for ABC importers and exporters are discussed.

[MEMBRANES] Activation of Phospholipase A2 by Ternary Model Membranes

Simonsen, A. C. — 2008-04-23

Formation of liquid-ordered domains in model membranes can be linked to raft formation in cellular membranes. The lipid stoichiometry has a governing influence on domain formation and consequently, biochemical hydrolysis of specific lipids has the potential to remodel domain features. Activation of phospholipase A₂ (PLA₂) by ternary model membranes with three components (DOPC/DPPC/Cholesterol) can potentially change the domain structure by preferential hydrolysis of the phospholipids. Using fluorescence microscopy, this work investigates the changes in domain features that occur upon PLA₂ activation by such ternary membranes. Double-supported membranes are used, which have minimal interactions with the solid support. For membranes prepared in the coexistence region, PLA₂ induces a decrease of the liquid-disordered (L_d) phase and an increase of the liquid-ordered (L_o) phase. A striking observation is that activation by a uniform membrane in the L_d phase leads to nucleation and growth of L_o-like domains. This phenomenon relies on the initial presence of cholesterol and no PLA₂ activation is observed by membranes purely in the L_o phase. The observations can be rationalized by mapping partially hydrolyzed islands onto trajectories in the phase diagram. It is proposed that DPPC is protected from hydrolysis through interactions with cholesterol, and possibly the formation of condensed complexes. This leads to specific trajectories which can account for the observed trends. The results demonstrate that PLA₂ activation by ternary membrane islands may change the global lipid composition and remodel domain features while preserving the overall membrane integrity.

[MEMBRANES] Specific Lipids Supply Critical Negative Spontaneous Curvature--An Essential Component of Native Ca2+-Triggered Membrane Fusion

2008-04-23

The Ca²⁺-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, T, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary "spontaneous" lipid rearrangements that must occur during native membrane fusion—Ca²⁺-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.

[MEMBRANES] Influence of the Lamellar Phase Unbinding Energy on the Relative Stability of Lamellar and Inverted Cubic Phases

Siegel, D. P., Tenchov, B. G. — 2008-04-23

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (Q_II) phases at temperatures between the lamellar (L) and inverted hexagonal (H_II) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct L/H_II phase transitions and may form intermediate Q_II phases only after the temperature is cycled repeatedly across the L/H_II phase transition temperature, T_H, or when the H_II phases are cooled from T > T_H. This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the L phase bilayers, g_u, that serves to stabilize the L phase relative to the nonlamellar phases. The planar L phase bilayers must separate for a Q_II phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of Q_II phase from L at temperatures near the L/Q_II transition temperature (T_Q). Using g_u and elastic constant values typical of unsaturated PEs, we show that g_u is sufficient to make T_Q > T_H for the latter lipids. Such systems would display direct L -> H_II transitions, and a Q_II phase might only form as a metastable phase upon cooling of the H_II phase. The g_u values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase T_Q by only a few degrees, consistent with observations of these systems. This influence of g_u also rationalizes the effect of some aqueous solutes to increase the rate of Q_II formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring T_Q and on measuring Q_II phase unit cell dimensions, respectively. In view of the effect of g_u on T_Q that we describe here, the latter method, which does not depend on the value of g_u, is preferable.

[MEMBRANES] Energetics of Hydrophobic Matching in Lipid-Protein Interactions

Marsh, D. — 2008-04-23

Lipid chain length modulates the activity of transmembrane proteins by mismatch between the hydrophobic span of the protein and that of the lipid membrane. Relative binding affinities of lipids with different chain lengths are used to estimate the excess free energy of lipid-protein interaction that arises from hydrophobic mismatch. For a wide range of integral proteins and peptides, the energy cost is much less than the elastic penalty of fully stretching or compressing the lipid chains to achieve complete hydrophobic matching. The chain length dependences of the free energies of lipid association are described by a model that combines elastic chain extension with a free energy term that depends linearly on the extent of residual mismatch. The excess free energy densities involved lie in the region of 0.5–2.0 k_BT.nm^–2. Values of this size could arise from exposure of hydrophobic groups to polar portions of the lipid or protein, but not directly to water, or alternatively from changes in tilt of the transmembrane helices that are energetically comparable to those activating mechanosensitive channels. The influence of hydrophobic mismatch on dimerization of transmembrane helices and their transfer between lipid vesicles, and on shifts in chain-melting transitions of lipid bilayers by incorporated proteins, is analyzed by using the same thermodynamic model. Segmental order parameters confirm that elastic lipid chain distortions are insufficient to compensate fully for the mismatch, but the dependence on chain length with tryptophan-anchored peptides requires that the free energy density of hydrophobic mismatch should increase with increasing extent of mismatch.

[MUSCLE AND CONTRACTILITY] Clockwise Translocation of Microtubules by Flagellar Inner-Arm Dyneins In Vitro

Kikushima, K., Kamiya, R. — 2008-04-23

Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.

[PHOTOBIOPHYSICS] Characterization of the Primary Photochemistry of Proteorhodopsin with Femtosecond Spectroscopy

2008-04-23

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp⁹⁷, is deprotonated) and at pH 6.5 (with Asp⁹⁷ protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of ~0.2 ps, ~2 ps, and ~20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerizaton pathways that lead to the K-state: one with an effective rate of ~(2 ps)^–1 and the other with a rate of ~(20 ps)^–1. At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of ~0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp²²⁷, which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.

[PROTEINS] Insights into Structure, Stability, and Toxicity of Monomeric and Aggregated Polyglutamine Models from Molecular Dynamics Simulations

Esposito, L., Paladino, A., Pedone, C., Vitagliano, L. — 2008-04-23

Nine genetically inherited neurodegenerative diseases are linked to abnormal expansions of a polyglutamine (polyQ) encoding region. Over the years, several structural models for polyQ regions have been proposed and confuted. The cross-β-spine steric zipper motif, identified recently for the GNNQQNY peptide, represents an attractive model for amyloid fibers formed by polyQ fragments. Here we report a detailed molecular dynamics investigation of polyQ models assembled by cross-β-spine steric zipper motifs. Our simulations indicate clearly that these assemblies are very stable. Glutamine side chains contribute strongly to the overall stability of the models by fitting perfectly within the zipper. In contrast to GNNQQNY zipper motifs, hydrogen bonding interactions provide a significant contribution to the overall stability of polyQ models. Molecular dynamics simulations carried out on monomeric polyQ forms (composed by 40–60 residues) show clearly that they can also assume structures stabilized by steric zipper motifs. Based on these findings, we build monomeric polyQ models that can explain recent data on the toxicity exerted by these species. In a more general context, our data suggests that polyQ models with interdigitated side chains can provide a structural rationale to several literature experiments on polyQ formation, stability, and toxicity.

[PROTEINS] The Kinetics of the Hydrogen/Deuterium Exchange of Epidermal Growth Factor Receptor Ligands

2008-04-23

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3₁₀-helix and random coil with varying contributions of β-sheets and β-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor- (TGF-), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min^–1 (Tyr), 0.09 min^–1 (Arg, β-turns), and 1.88 x 10^–3 min^–1 (β-sheets and 3₁₀-helix); and for TGF- 0.91 min^–1 (Tyr), 0.27 min^–1 (Arg, β-turns), and 1.41 x 10^–4 min^–1 (β-sheets). The time constants for AR 0.47 min^–1 (Tyr), 0.04 min^–1 (Arg), and 1.00 x 10^–4 min^–1 (buried 3₁₀-helix, β-turns, and β-sheets); for HB-EGF 0.89 min^–1 (Tyr), 0.14 min^–1 (Arg and 3₁₀-helix), and 1.00 x 10^–3 min^–1 (buried 3₁₀-helix, β-sheets, and β-turns); and for epiregulin 0.16 min^–1 (Tyr), 0.03 min^–1 (Arg), and 1.00 x 10^–4 min^–1 (3₁₀-helix and β-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.

[PROTEINS] Distinct Unfolding and Refolding Pathways of Ribonuclease A Revealed by Heating and Cooling Temperature Jumps

2008-04-23

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (H^#), and activation entropy (S^#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative H^# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro¹¹⁴ isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp¹¹⁵. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.

[PROTEINS] Spectroscopic, Structural, and Functional Characterization of the Alternative Low-Spin State of Horse Heart Cytochrome c

2008-04-23

The alternative low-spin states of Fe³⁺ and Fe²⁺ cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met⁸⁰ by another strong field ligand at the sixth heme iron coordination position, Fe³⁺ ALSScytc exhibited 1-nm Soret band blue shift and enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe³⁺ and Fe²⁺ ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp⁵⁹ fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His³³ and His²⁶ carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His³³ replaced by Asn) pointing out Lys⁷⁹ as the probable heme iron ligand. Fe³⁺ ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential ~200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.

[PROTEINS] Remote Mutations and Active Site Dynamics Correlate with Catalytic Properties of Purine Nucleoside Phosphorylase

Saen-Oon, S., Ghanem, M., Schramm, V. L., Schwartz, S. D. — 2008-04-23

It has been found that with mutation of two surface residues (Lys²² -> Glu and His¹⁰⁴ -> Arg) in human purine nucleoside phosphorylase (hPNP), there is an enhancement of catalytic activity in the chemical step. This is true although the mutations are quite remote from the active site, and there are no significant changes in crystallographic structure between the wild-type and mutant active sites. We propose that dynamic coupling from the remote residues to the catalytic site may play a role in catalysis, and it is this alteration in dynamics that causes an increase in the chemical step rate. Computational results indicate that the mutant exhibits stronger coupling between promotion of vibrations and the reaction coordinate than that found in native hPNP. Power spectra comparing native and mutant proteins show a correlation between the vibrations of Immucillin-G (ImmG):O5'···ImmG:N4' and H257:N···ImmG:O5' consistent with a coupling of these motions. These modes are linked to the protein promoting vibrations. Stronger coupling of motions to the reaction coordinate increases the probability of reaching the transition state and thus lowers the activation free energy. This motion has been shown to contribute to catalysis. Coincident with the approach to the transition state, the sum of the distances of ImmG:O4'···ImmG:O5'···H257:N became smaller, stabilizing the oxacarbenium ion formed at the transition state. Combined results from crystallography, mutational analysis, chemical kinetics, and computational analysis are consistent with dynamic compression playing a significant role in forming the transition state. Stronger coupling of these pairs is observed in the catalytically enhanced mutant enzyme. That motion and catalysis are enhanced by mutations remote from the catalytic site implicates dynamic coupling through the protein architecture as a component of catalysis in hPNP.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Imaging Single Virus Particles on the Surface of Cell Membranes by High-Resolution Scanning Surface Confocal Microscopy

2008-04-23

We have developed a high-resolution scanning surface confocal microscopy technique capable of imaging single virus-like particles (VLPs) on the surfaces of cells topographically and by fluorescence. The technique combines recently published single-molecule-resolution ion-conductance microscopy that acquires topographical data with confocal microscopy providing simultaneous fluorescent imaging. In our experiments we have demonstrated that the cell membrane exhibits numerous submicrometer-sized surface structures that could be topographically confused with virus particles. However, simultaneous acquisition of confocal images allows the positions of fluorescently tagged particles to be identified. Using this technique, we have, for the first time, visualized single polyoma VLPs adsorbed onto the cell membrane. Observed VLPs had a mean width of 108 ± 16 nm. The particles were randomly distributed across the cell membrane, and no specific interactions were seen with cell membrane structures such as microvilli. These experiments demonstrate the utility of this new microscope for imaging the interactions of nanoparticles with the cell surface to provide novel insights into the earliest interactions of viruses and other nanoparticles such as gene therapy vectors with the cell.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Label-Free Calcium Imaging in Ischemic Retinal Tissue by TOF-SIMS

2008-04-23

The distribution and movement of elemental ions in biologic tissues is critical for many cellular processes. In contrast to chemical techniques for imaging the intracellular distribution of ions, however, techniques for imaging the distribution of ions across tissues are not well developed. We used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to obtain nonlabeled high-resolution analytic images of ion distribution in ischemic retinal tissues. Marked changes in Ca²⁺ distribution, compared with other fundamental ions, such as Na⁺, K⁺, and Mg²⁺, were detected during the progression of ischemia. Furthermore, the Ca²⁺ redistribution pattern correlated closely with TUNEL-positive (positive for terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labeling) cell death in ischemic retinas. After treatment with a calcium chelator, Ca²⁺ ion redistribution was delayed, resulting in a decrease in TUNEL-positive cells. These results indicate that ischemia-induced Ca²⁺ redistribution within retinal tissues is associated with the order of apoptotic cell death, which possibly explains the different susceptibility of various types of retinal cells to ischemia. Thus, the TOF-SIMS technique provides a tool for the study of intercellular communication by Ca²⁺ ion movement.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Dark States in Monomeric Red Fluorescent Proteins Studied by Fluorescence Correlation and Single Molecule Spectroscopy

Hendrix, J., Flors, C., Dedecker, P., Hofkens, J., Engelborghs, Y. — 2008-04-23

Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins, and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry, and mStrawberry by means of fluorescence correlation spectroscopy and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a "loose" conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-color fluorescence correlation spectroscopy inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well separated.

[CELL BIOPHYSICS] How Cells Tiptoe on Adhesive Surfaces before Sticking

Pierres, A., Benoliel, A.-M., Touchard, D., Bongrand, P. — 2008-04-23

Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5–10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30–40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension.

[CELL BIOPHYSICS] TGF{beta}/Activin/Nodal Pathway in Inhibition of Human Embryonic Stem Cell Differentiation by Mechanical Strain

Saha, S., Ji, L., de Pablo, J. J., Palecek, S. P. — 2008-04-23

Cyclic biaxial mechanical strain has been reported to inhibit human embryonic stem cell differentiation without selecting against survival of differentiated or undifferentiated cells. We show that TGFβ/Activin/Nodal signaling plays a crucial role in repression of human embryonic stem cell (hESC) differentiation under mechanical strain. Strain-induced transcription of TGFβ1, Activin A, and Nodal, and upregulated Similar to Mothers Against Decapentaplegic homolog (Smad)2/3 phosphorylation in undifferentiated hESC. TGFβ/Activin/Nodal receptor inhibitor SB431542 stimulated differentiation of hESCs cultured under biaxial strain. Exogenous addition of TGFβ1, Activin A, or Nodal alone was insufficient to stimulate hESC self-renewal to replicate behavior of hESCs in presence of strain. However, exogenous TGFβ1 and Activin A in combination partially replicated the self-renewing phenotype induced by strain but when combined with strain did not further stimulate self-renewal. In presence of mechanical strain, addition of a neutralizing antibody to TGFβ1 promoted hESC differentiation whereas inhibition of Activin A by Follistatin promoted hESC differentiation to a lesser extent. Together, these findings show that TGFβ superfamily activation of Smad2/3 is required for repression of spontaneous differentiation under strain and suggest that strain may induce autocrine or paracrine signaling through TGFβ superfamily ligands.

[CELL BIOPHYSICS] Fluctuations of the Red Blood Cell Membrane: Relation to Mechanical Properties and Lack of ATP Dependence

Evans, J., Gratzer, W., Mohandas, N., Parker, K., Sleep, J. — 2008-04-23

We have analyzed the fluctuations of the red blood cell membrane in both the temporal (((s^–1)) and spatial (q(m^–1)) frequency domains. The cells were examined over a range of osmolarities leading to cell volumes from 50% to 170% of that in the isotonic state. The fluctuations of the isotonic cell showed an ~q^–3-dependence, indicative of a motion dominated by bending, with an inferred bending modulus of ~9 x 10^–19J. When the cells were osmotically swollen to just below the point of lysis (166% of physiological volume), a q^–1-dependence of the fluctuations supervened, implying that the motion was now dominated by membrane tension; estimated as ~1.3 x 10^–4 nm^–1. When, on the other hand, the cells were osmotically dehydrated, the fluctuation amplitude progressively decreased. This was caused by a rise in internal viscosity, as shown by measurements on resealed ghosts containing a reduced hemoglobin concentration, which displayed no such effect. We examined, in addition, cells depleted of ATP, before the onset of echinocytosis, and could observe no change in fluctuation amplitude. We conclude that the membrane fluctuations of the red cell are governed by bending modulus, membrane tension, and cytosolic viscosity, with little or no dependence on the presence or absence of ATP.

[OTHER] Intermolecular Association Provides Specific Optical and NMR Signatures for Serotonin at Intravesicular Concentrations

Nag, S., Balaji, J., Madhu, P. K., Maiti, S. — 2008-04-23

Neurotransmitter vesicles contain biomolecules at extraordinarily high concentrations (hundreds of millimoles/liter). Such concentrations can drive intermolecular associations, which may affect vesicular osmolarity and neuronal signaling. Here we investigate whether aqueous serotonin (a monoamine neurotransmitter) forms oligomers at intravesicular concentrations and whether these oligomers have specific spectroscopic signatures that can potentially be used for monitoring neuronal storage and release. We report that, as serotonin concentration is increased from 60 µM to 600 mM, the normalized fluorescence spectrum of serotonin displays a growing long-wavelength tail, with an isoemissive point at 376 nm. The fluorescence decay is monoexponential with a lifetime of 4 ns at low concentrations but is multiexponential with an average lifetime of 0.41 ns at 600 mM. A 600 mM serotonin solution has 30% less osmolarity than expected for monomeric serotonin, indicating oligomer formation. The proton NMR chemical shifts move upfield by as much as 0.3 ppm at 600 mM compared to those at 10 mM, indicating a stacking of the serotonin indole moieties. However, no intermolecular crosspeak is evident in the two-dimensional NMR rotating frame Overhauser effect spectroscopy spectrum even at 600 mM, suggesting that oligomeric structures are possibly weakly coupled. The appearance of a single peak for each proton suggests that the rate of interconversion between the monomeric and the oligomeric structures is faster than 240 Hz. A stopped-flow kinetic experiment also confirms that the rate of dissociation is faster than 100 ms. We conclude that serotonin forms oligomers at intravesicular concentrations but becomes monomeric quickly on dilution. NMR signatures of the oligomers provide potential contrast agents for monitoring the activity of serotonergic neurons in vivo.

[CORRECTION] Correction:

2008-04-23