Biophysical Journal BioFAST

[BIOPHYSICAL LETTERS] Instantaneous Amyloid Fibril Formation of {alpha}-Synuclein from the Oligomeric Granular Structures in the Presence of Hexane

Lee, J., Bhak, G., Lee, S.-G., Paik, S. R. — 2008-05-09

Amyloid fibrils found in various neurodegenerative disorders are also recognized as high-performance protein nanomaterials with a formidable rigidity. Elucidation of an underlying molecular mechanism of the amyloid fibril formation is crucial not only to develop controlling strategy toward the diseases, but also to apply the protein fibrils for future nanobiotechnology. -Synuclein is an amyloidogenic protein responsible for the radiating filament formation within Lewy bodies of Parkinson's disease (PD). The amyloid fibril formation of -synuclein has been demonstrated to be induced from the oligomeric granular species of the protein acting as a growing unit by experiencing structural rearrangement within the preformed oligomeric structures in the presence of an organic solvent of hexane. This granule-based concerted amyloid fibril formation model would parallel the prevalent notion of nucleation-dependent fibrillation mechanism in the area of amyloidosis.

[CELL BIOPHYSICS] New and Notable for Ca2+-mobility in the sarcoplasmic reticulum of ventricular myocytes is low

Smith, G. L, MacQuaide, N. — 2008-05-09

None

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Effect of Clotrimazole on the Pump Cycle of the Na,K-ATPase

2008-05-09

The effect of the antimycotic Clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady state fluorescence experiments with the electrochromic styryl dye RH421, were combined with those achieved by a pre-steady method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable to monitor the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E₂ state, E₂^CLT(X₂) that acts as a "dead-end" side track of the pump cycle, where X stands for H⁺ or K⁺. Na⁺ binding, enzyme phosphorylation, and Na⁺ transport were not affected by CLT, and at high CLT concentrations about one third of the enzyme remained active in the physiological transport mode. The presence of Na⁺ and K⁺ destabilized the inactivated form of the Na,K-ATPase.

[BIOPHYSICAL THEORY AND MODELING] REACH Coarse-Grained Biomolecular Simulation: Transferability between Different Protein Structural Classes

Moritsugu, K., Smith, J. C. — 2008-05-09

Coarse graining of protein interactions provides a means of simulating large biological systems. The REACH coarse-graining method, in which the force constants of a residue-scale elastic network model are calculated from the variance-covariance matrix obtained from atomistic molecular dynamics (MD) simulation, involves direct mapping between scales without the need for iterative optimization. Here, the transferability of the REACH force field is examined between protein molecules of different structural classes. As test cases, myoglobin (all ), plastocyanin (all β) and dihydrofolate reductase (/β) are taken. The force constants derived are found to be closely similar in all three proteins. A MD version of REACH is presented, and low-temperature coarse-grained REACH MD simulations of the three proteins compared with atomistic MD results. The mean-square fluctuations of the atomistic MD are well reproduced by the coarse-grained MD. Model functions for the coarse-grained interactions, derived by averaging over the three proteins, are also shown to produce fluctuations in good agreement with the atomistic MD. The results indicate that, similarly to the usage of atomistic force fields, it is now possible to use a single, generic REACH force field for all protein studies, without having first to derive parameters from atomistic MD simulation for each individual system studied. The REACH method is thus likely to be a reliable way of determining spatiotemporal motion of a variety of proteins without the need for expensive computation of long atomistic MD simulations.

[BIOPHYSICAL THEORY AND MODELING] RANDOM-WALK MODEL OF DIFFUSION IN 3-DIMENSIONS IN BRAIN EXTRACELLULAR SPACE: COMPARISON WITH MICROFIBEROPTIC PHOTOBLEACHING MEASUREMENTS

Jin, S., Zador, Z., Verkman, A. S. — 2008-05-09

Diffusion through the extracellular space (ECS) in brain is important in drug delivery, intercellular communication, and extracellular ionic buffering. The ECS comprises ~20% of brain parenchymal volume and contains cell-cell gaps ~50 nm. We developed a random-walk model to simulate macromolecule diffusion in brain ECS in 3-dimensions using realistic ECS dimensions. Model inputs included ECS volume fraction (), cell size, cell-cell gap geometry, intercellular 'lake' (expanded regions of brain ECS) dimensions, and molecular size of the diffusing solute. Model output was relative solute diffusion in water vs. brain ECS (D_o/D). Experimental D_o/D for comparison with model predictions was measured using a microfiberoptic fluorescence photobleaching method involving stereotaxic insertion of a micron-size optical fiber into mouse brain. D_o/D for the small solute calcein in different regions of brain was in the range 3.0-4.1, and increased with brain cell swelling following water intoxication. D_o/D also increased with increasing size of the diffusing solute, particularly in deep brain nuclei. Simulations of measured D_o/D using realistic , cell size and cell-cell gap required the presence of intercellular 'lakes' at multi-cell contact points, and the contact length of cell-cell gaps to be least 50-fold smaller than cell size. The model accurately predicted Do/D for different solute sizes. Also, the modeling revealed unanticipated effects on D_o/D of changing ECS and cell dimensions that implicated 'solute trapping' by lakes. Our model establishes the geometric constraints to account quantitatively for the relatively modest slowing of solute and macromolecule diffusion in brain ECS.

[MEMBRANES] PrP(106-126) does not interact with membranes under physiological conditions

Henriques, S. T., Pattenden, L. K., Aguilar, M. I., Castanho, M. A.R.B. — 2008-05-09

Transmissible spongiform encephalopathies are neurodegenerative diseases with characteristic accumulation of an abnormal isoform of the prion protein, PrP^Sc. Its fragment 106-126 was reported to maintain most of the pathological features of PrP^Sc and a role in neurodegeneration was proposed based on the modulation of membrane properties and channel formation. While the ability of PrP^Sc to modulate membranes and/or form channels in membranes has not been clearly demonstrated, if these processes are important, peptide-membrane interactions would be a key feature to the toxicity of PrP^Sc. In the present work, the interaction of PrP(106-126) with model membranes comprising typical lipid identities as well as more specialised lipids such as phosphatidylserine and GM1 ganglioside, was examined using surface plasmon resonance and fluorescence methodologies. This comprehensive study examines different parameters relevant to characterisation of peptide-membrane interactions including: membrane charge, viscosity, lipid composition, pH and ionic strength. We report that PrP(106-126) has a low affinity for lipid membranes under physiological conditions without evidence for membrane disturbances. Membrane insertion and leakage only occurs under conditions where strong electrostatic interactions operate. These results support the hypothesis that the physiological prion protein, PrP^C, mediates PrP(106-126) toxic effects in neuronal cells.

[MEMBRANES] Kinetics, Statistics, and Energetics of Lipid Membrane Electroporation Studied by Molecular Dynamics Simulations

Bockmann, R. A, de Groot, B. L, Kakorin, S., Neumann, E., Grubmuller, H. — 2008-05-09

Membrane electroporation is the method to directly transfer bioactive substances such as drugs and genes into living cells, as well as preceding electrofusion. Although much information on the microscopic mechanism has been obtained both from experiment and simulation, the existence and nature of possible intermediates is still unclear. To elucidate intermediates of electropore formation by direct comparison with measured prepore formation kinetics, we have carried out 49 atomistic electroporation simulations on a POPC bilayer for electric field strengths between 0.04 and 0.7 V/nm. A statistical theory is developed to facilitate direct comparison of experimental (macroscopic) prepore formation kinetics with the (single event) preporation times derived from the simulations, which also allows to extract an effective number of lipids involved in each pore formation event. A linear dependency of the activation energy for prepore formation on the applied field is seen, with quantitative agreement between experiment and simulation. The distribution of preporation times suggests a four state pore formation model. The model involves a first intermediate characterized by a differential tilt of the polar lipid head groups on both leaflets, and a second intermediate ('prepore'), where a polar chain across the bilayer is formed by 3-4 lipid head groups and several water molecules, thereby providing a microscopic explanation for the polarizable volume derived previously from the measured kinetics. An average pore radius of 0.47 ± 0.15 nm is seen, in favourable agreement with conductance measurements and electrooptical experiments of lipid vesicles.

[PROTEINS] Insights into Stability and Toxicity of Amyloid-Like Oligomers by Replica Exchange Molecular Dynamics Analyses

De Simone, A., Esposito, L., Pedone, C., Vitagliano, L. — 2008-05-09

Deposition of insoluble amyloid plaques is frequently associated with a large variety of neurodegenerative diseases. However, data collected in the last decade have suggested that the neurotoxic action is exerted by pre-fibrillar, soluble assemblies of amyloid-forming proteins and peptides. The scarcity of structural data available for both amyloid-like fibrils and soluble oligomers is a major limitation for the definition of the molecular mechanisms linked to the onset of these diseases. Recently, the structural characterization of GNNQQNY and other peptides has revealed a general feature of amyloid-like fibers, the so-called steric zipper motif. However, still very little is known about the prefibrillar oligomeric forms. By using replica exchange molecular dynamics (REMD) we carried out extensive analyses of the properties of several small and medium GNNQQNY aggregates arranged through the steric zipper motif. Our data show that the assembly formed by two sheets, each made of two strands, arranged as in the crystalline states are highly unstable. Conformational free energy surfaces indicate that the instability of the model can be ascribed to the high reactivity of edge backbone hydrogen bonding donors/acceptors. On the other hand, data on larger models show that steric zipper interactions may keep small oligomeric forms in a stable state. These models simultaneously display two peculiar structural motifs: a tightly packed steric zipper interface and a large number of potentially reactive exposed strands. The presence of highly reactive groups on these assemblies likely generates two distinct evolutions. On one side the reactive groups quickly lead, through self-association, to the formation of ordered fibrils, on the other they may interfere with several cellular components thereby generating toxic effects. In this scenario, fiber formation propensity and toxicity of oligomeric states are two different manifestations of the same property: the hyper-reactivity of the exposed strands.

[PHOTOBIOPHYSICS] Balance between ultrafast parallel reactions in the Green Fluorescent Protein has a structural origin

van Thor, J. J, Ronayne, K. L., Towrie, M., Sage, J T. — 2008-05-09

The fluorescence photocycle of the Green Fluorescent Protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein sidechain conformation of Glutamate 222 and reactivity is established, particularly of the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin and we report on the vibrational properties of responsive aminoacids on an ultrafast time-scale. Blue excitation of GFP drives two parallel excited state deuteron transfer reactions (ESPT) with 10 ps and 75 ps time-constants to the buried carboxylic acid sidechain of Glutamate 222 via a hydrogen bonding network. Assignment of 1456 cm^-1 and 1441 cm^-1 modes to _sym and 1564 cm^-1 and 1570 cm^-1 features to _asym of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant, and consistent with photoselection measurements. In contrast to the wild type, measurements of E222D can be described with only one difference spectrum, with the _sym mode at 1435 cm^-1 and the _asym mode at 1567 cm^-1, also correlating a large _asym-sym with slow ESPT kinetics. DFT calculations and published model compound and theoretical studies relate differences in _asym-sym to the strength and number of hydrogen-bonding interactions that is detected via equilibrium geometry and COO^- stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall ESPT process, consistent with recent theoretical predictions. Photoselection measurements show agreement for localised C=O vibrations of chromophore, Q69 and E222 with DFT and ab initio calculations placed in the X-ray geometry and provide their vibrational response in the intermediates in the photocycle

[CELL BIOPHYSICS] Tightly-Regulated and Heritable Division Control in Single Bacterial Cells

Siegal-Gaskins, D., Crosson, S. — 2008-05-09

The robust surface adherence property of the aquatic bacterium, Caulobacter crescentus, permits visualization of single cells in a linear microfluidic culture chamber over an extended number of generations. The division rate of Caulobacter in this continuous-flow culture environment is substantially faster than in other culture apparati and is independent of flow velocity. Analysis of the growth and division of single, isogenic cells reveals that the cell cycle control network of this bacterium generates an oscillatory output with a coefficient of variation that is lower than that of all other bacterial species measured to date. DivJ, a regulator of polar cell development, is necessary for maintaining low variance in interdivision timing, as transposon disruption of divJ significantly increases the coefficient of variation of both interdivision time and the rate of cell elongation. Moreover, interdivision time and cell division arrest are significantly correlated between mother and daughter cells, providing evidence for epigenetic inheritance of cell division behavior in Caulobacter. The single cell growth/division results reported here suggest that future predictive models of Caulobacter cell cycle regulation should include parameters describing the variance and inheritance properties of this system.

[CELL BIOPHYSICS] Modulation of SR Ca Release by Luminal Ca and Calsequestrin in Cardiac Myocytes: Effects of CASQ2 Mutations Linked to Sudden Cardiac Death

2008-05-09

Cardiac calsequestrin (CASQ2) is an intra-sarcoplasmic reticulum (SR) low-affinity Ca-binding protein, mutations in which are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2^DEL) or CASQ2 containing a point mutation (CASQ2^R33Q), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer (FRET) in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2^DEL or CASQ2^R33Q altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2^DEL disrupted the CASQ2 polymerization required for high capacity Ca binding, while CASQ2^R33Q compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild type CASQ2 and CASQ2^DEL –expressing myocytes, suggesting that a decline in [Ca]_SR is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2^R33Q markedly lowered the [Ca]_SR threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, both lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.

[BIOPHYSICAL THEORY AND MODELING] Representation of Collective Electrical Behavior Of Cardiac Cell Sheets

Weinberg, S., Iravanian, S., Tung, L. — 2008-05-09

The electrocardiogram (ECG) is a measure of the collective electrical behavior of the heart based on body surface measurements. With computational models or tissue preparations, various methods have been used to compute the pseudo-ECG (pECG) of bipolar and unipolar leads that can be given clinical interpretation. When spatial maps of transmembrane potential (Vm) are available, pECG can be derived from a weighted sum of the spatial gradients of Vm. The concept of a lead field can be used to define sensitivity curves for different bipolar and unipolar leads and to determine an effective operating height for the bipolar lead position for a 2-D sheet of heart cells. The pseudo-vectorcardiogram (pVCG) is computed from orthogonal bipolar lead voltages, which are derived in this study from optical voltage maps of cultured monolayers of cardiac cells. Rate and propagation direction for paced activity, rotation frequency for reentrant activity, direction of the common pathway for figure-eight reentry, and transitions from paced activity to reentry can all be distinguished using the pVCG. In contrast, the unipolar pECG does not clearly distinguish among many of the different types of electrical activity. We also show that pECG can be rapidly computed by two geometrically weighted sums of Vm, one that is summed over the area of the cell sheet and the other over the perimeter of the cell sheet. Our results are compared with those of an ad-hoc difference method used in the past that consists of a simple difference of the sum of transmembrane potentials on one side of a tissue sheet and that of the other.

[BIOPHYSICAL THEORY AND MODELING] Simulated de novo assembly of Golgi compartments by selective cargo capture during vesicle budding and targeted vesicle fusion

Gong, H., Sengupta, D., Linstedt, A. D., Schwartz, R. — 2008-05-09

The Golgi apparatus is comprised of stacked cisternal membranes forming subcompartments specialized for post-translational processing of newly synthesized secretory cargo. Recent experimental evidence indicates that the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum but the mechanism by which the membranes self assemble into compartmentalized structures remains unknown. We developed a discrete-event computer simulation model to test whether two fundamental mechanisms- vesicle coat mediated selective concentration of SNARE proteins during vesicle formation and SNARE-mediated selective fusion of vesicles- suffice to generate and maintain compartments. Simulations verified that this minimal model is adequate for homeostasis of pre-established compartments, even in response to small perturbations, and for de novo formation of stable compartments. The model led to a novel prediction that Golgi size is, in part, dependent on target SNARE expression level. This prediction was supported by a demonstration that exogenous expression of the Golgi target SNARE syntaxin-5 alters Golgi size in living cells.

[OTHER] Timing and dynamics of single cell gene expression in the arabinose utilization system

Megerle, J. A., Fritz, G., Gerland, U., Jung, K., Radler, J. O. — 2008-05-09

The arabinose utilization system of E. coli displays a stochastic "all or nothing" response at intermediate levels of arabinose, where the population divides into a fraction catabolizing the sugar at a high rate (ON state) and a fraction not utilizing arabinose (OFF state). Here we study this decision process in individual cells, focusing on the dynamics of the transition from the OFF to the ON state. Using quantitative time-lapse microscopy, we determine the time delay between inducer addition and fluorescence onset of a GFP reporter. Through independent characterization of the GFP maturation process, we can separate the lag time caused by the reporter from the intrinsic activation time of the arabinose system. The resulting distribution of intrinsic time delays scales inversely with the external arabinose concentration, and is compatible with a simple stochastic model for arabinose uptake. Our findings support the idea that the heterogeneous timing of gene induction is causally related to a broad distribution of uptake proteins at the time of sugar addition.

[BIOPHYSICAL THEORY AND MODELING] The Interaction of Phospholipase A2 with a Phospholipid Bilayer: Coarse-Grained Molecular Dynamics Simulations

Wee, C.-L., Balali-Mood, K., Gavaghan, D., Sansom, M. S.P. — 2008-05-09

A number of membrane-active enzymes act in a complex environment formed by the interface between a lipid bilayer and bulk water. Whilst X-ray diffraction studies yield structures of isolated enzyme molecules, a detailed characterisation of their interactions with the interface requires a measure of how deeply such a membrane-associated protein penetrates into a lipid bilayer. Here, we apply coarse-grained molecular dynamics (CG-MD) simulations to probe the interaction of porcine pancreatic phospholipase A2 (PLA2) with a lipid bilayer containing palmitoyl-oleoyl-phosphatidyl choline (POPC) and palmitoyl-oleoyl-phosphatidyl glycerol (POPG) molecules. We also used a configuration from a CG-MD trajectory to initiate two atomistic MD (AT-MD) simulations. The results of the CG and AT simulations are evaluated by comparison with available experimental data. The membrane-binding surface of PLA2 consists of a patch of hydrophobic residues surrounded by polar and basic residues. We show this proposed footprint interacts preferentially with the anionic headgroups of the POPG molecules. Thus, both electrostatic and hydrophobic interactions determine the location of PLA2 relative to the bilayer. From a general perspective, this study demonstrates that CG-MD simulations may be used to reveal the orientation and location of a membrane-surface bound protein relative to a lipid bilayer, which may subsequently be refined by AT-MD simulations to probe more detailed interactions.

[BIOPHYSICAL THEORY AND MODELING] Towards resolution of ambiguity for the unfolded state

Beaucage, G. — 2008-05-09

The unfolded states in proteins and nucleic acids remain weakly understood despite their importance to understanding folding processes; misfolding diseases (Parkinson's & Alzheimer's); natively unfolded proteins (as many as 30% of eukaryotic proteins [Fink, 2005]); and to the study of ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is proposed to quantify the molar degree of folding and the unfolded state. The model takes a global view of protein structure and can be applied to a number of analytic methods and to simulations. Three examples are given of application to small-angle scattering from pressure induced unfolding of SNase, from acid unfolded Cyt c and from folding of Azoarcus ribozyme. These examples quantitatively show 3 characteristic unfolded states for proteins, the statistical nature of a protein folding pathway and the relationship between extent of folding and chain size during folding for charge driven folding in RNA.

[MEMBRANES] Factors Influencing Local Membrane Curvature Induction by N-BAR Domains as Revealed by Molecular Dynamics Simulations

Blood, P. D, Swenson, R. D., Voth, G. A. — 2008-05-09

N-BAR domains are protein modules that bind to and induce curvature in membranes via a charged concave surface and N-terminal amphipathic helices. Recently, molecular dynamics simulations have demonstrated that the N-BAR domain can induce a strong local curvature that matches the curvature of the BAR domain surface facing the bilayer. Here we present further molecular dynamics simulations that examine in greater detail the roles of the concave surface and amphipathic helices in driving local membrane curvature. We find that the strong curvature induction observed in our previous simulations requires the stable presentation of the charged concave surface to the membrane and is not driven by the membrane-embedded amphipathic helices. Nevertheless, without these amphipathic helices embedded in the membrane, the N-BAR domain does not maintain a close association with the bilayer, and fails to drive membrane curvature. Increasing the membrane negative charge through the addition of PIP₂ facilitates closer association with the membrane in the absence of embedded helices. At sufficiently high concentrations, amphipathic helices embedded in the membrane drive membrane curvature independently of the BAR domain.

[BIOPHYSICAL THEORY AND MODELING] Positive Receptor Feedback during Lineage Commitment Can Generate Ultrasensitivity to Ligand and Confer Robustness to a Bistable Switch

Palani, S., Sarkar, C. A. — 2008-05-09

Cytokines and lineage-specific transcription factors are critical molecular effectors for terminal differentiation during hematopoiesis. Intrinsic transcription factor activity is often believed to drive commitment and differentiation, whereas cytokine receptor signals have been implicated in the regulation of cell proliferation, survival, and differentiation. In erythropoiesis, recent experimental findings provide direct evidence that erythropoietin (Epo) can generate commitment cues via the erythropoietin receptor (EpoR); specifically, EpoR signaling leads to activation of the transcription factor GATA-1, which then triggers transcription of erythrocyte-specific genes. In particular, activated GATA-1 induces two positive feedback loops in the system through the enhanced expression of both inactive GATA-1 and EpoR, the latter of which is externally regulatable by Epo. Based upon this network architecture, we present a mathematical model of GATA-1 activation by EpoR, which bidirectionally links a lineage-specific receptor and transcription factor. Our deterministic model offers insight into stimulus-response relationships between Epo and several downstream effectors. In addition to the survival signals that EpoR provides, steady-state analysis of our model suggests that receptor upregulation during lineage commitment can also generate ultrasensitivity to Epo and bistability in GATA-1 activity. These system-level properties can induce a switch-like characteristic during differentiation and provide robustness to the mature state. The topology also suggests a novel mechanism for achieving robust bistability in a purely deterministic manner without molecular cooperativity. The analytical solution of a generalized, minimal model is provided and the significance of each of the two positive feedback loops is elucidated through bifurcation analysis. This network topology, or variations thereof, may link other receptor-transcription factor pairs and may therefore be of general relevance in cellular decision-making.

[BIOPHYSICAL THEORY AND MODELING] Collective swimming and the dynamics of bacterial turbulence

Wolgemuth, C. — 2008-05-09

To swim, a bacterium pushes against the fluid within which it is immersed, generating fluid flow that dies off on a length scale comparable to the size of the bacterium. However, in dense colonies of bacteria, the bacteria are close enough that flow generated by swimming is substantial. For these cases, complex flows can arise due to the interaction and feedback between the bacteria and the fluid. Recent experiments on dense populations of swimming Bacillus subtilis have revealed a volume fraction-dependent transition from random swimming to transient jet and vortex patterns in the bacteria/fluid mixture. The fluid motions that are observed are reminiscent of flows that are observed around translating objects at moderate to high Reynolds numbers. In this paper, I present a two-phase model for the bacterial/fluid mixture. The model explains turbulent flows in terms of the dipole stress that the bacteria exert on the fluid, entropic elasticity due to the rod-shape of each bacterium, and the torque on the bacteria due to fluid gradients. Solving the equations in two-dimensions using realistic parameters, the model reproduces empirically-observed velocity fields. Dimensional analysis provides scaling relations for the dependence of the characteristic scales on the model parameters.

[MUSCLE AND CONTRACTILITY] Magnitude of sarcomere extension correlates with initial sarcomere length during lengthening of activated single fibers from soleus muscle of rats

Panchangam, A., Claflin, D. R., Palmer, M. L., Faulkner, J. A. — 2008-05-09

A laser-diffraction technique was developed that reported rapidly the lengths of sarcomeres (L_s) in serially-connected sectors of permeabilized single fibers. The apparatus translated a laser beam along the entire length of a fiber segment within 2 ms, with brief stops at each of 20 contiguous sectors. We tested the hypothesis that during lengthening contractions, when maximally activated fibers are stretched, sectors that contain the longer sarcomeres undergo greater increases in L_s than those containing shorter sarcomeres. Fibers (n = 16) were obtained from soleus muscles of adult male rats and middle portions, length 1.05 ± 0.11 mm (mean ± SD), were investigated. Single stretches of strain 27% and a strain rate of 54% s^-1 were initiated at maximum isometric stress and resulted in a 19 ± 9 % loss in isometric stress. The data on L_s revealed that: (1) the stretch was not distributed uniformly among the sectors, and (2) during the stretch, sectors at long L_s prior to the stretch elongated more than those at short lengths. The findings support the hypothesis that during stretches of maximally-activated skeletal muscles, sarcomeres at longer lengths are more susceptible to damage by excessive strain.

[BIOPHYSICAL THEORY AND MODELING] Molecular dynamics studies of polyethylene oxide and polyethylene glycol: Hydrodynamic radius and shape anisotropy

Lee, H., Venable, R. M., MacKerell, Jr., A. D., Pastor, R. W. — 2008-05-02

A revision (C35r) to the CHARMM ether force field is shown to reproduce experimentally observed conformational populations of dimethoxyethane (DME). Molecular dynamics (MD) simulations of 9, 18, 27, and 36-mers of polyethylene oxide (PEO) and 27-mers of polyethylene glycol (PEG) in water based on C35r yield a persistence length = 3.7 Å, in quantitative agreement with experimentally obtained values of 3.7 Å for PEO and 3.8 Å for PEG; agreement with experimental values for hydrodynamic radii of comparably sized PEG is also excellent. The exponent relating the radius of gyration and molecular weight (R_h M_w) of PEO from the simulations equals 0.515 ± 0.023, consistent with experimental observations that low molecular weight PEG behaves as an ideal chain. The shape anisotropy of hydrated PEO is 2.59:1.44:1.00. The dimension of the middle length for each of the polymers nearly equals the hydrodynamic radius R_h obtained from diffusion measurements in solution. This explains the correspondence of R_h and R_p, the pore radius of membrane channels: a polymer such as PEG diffuses with its long axis parallel to the membrane channel, and passes through the channel without substantial distortion.

[PROTEINS] Sequence-specific conformational flexibility of SNARE transmembrane helices probed by hydrogen/deuterium exchange

Stelzer, W., Poschner, B., Stalz, H., Heck, A., Langosch, D. — 2008-05-02

SNARE proteins mediate fusion of intracellular eukaryotic membranes and their -helical transmembrane domains are known to contribute to lipid bilayer mixing. Synthetic transmembrane domain peptides were previously shown to mimic the function of SNARE proteins in that they trigger liposome fusion in a sequence-specific fashion. Here, we performed a detailed investigation of the conformational dynamics of the transmembrane helices of the presynaptic SNAREs synaptobrevin II and syntaxin 1a. To this end, we recorded deuterium/hydrogen-exchange kinetics in isotropic solution as well as in the membrane-embedded state. In solution, the exchange kinetics of each peptide can be described by three different classes of amide deuteriums that exchange with different rate constants. These are likely to originate from exchange at different domains of the helices. Interestingly, the rate constants of each class vary with the TMD sequence. Thus, the exchange rate is position-specific and sequence-specific. Further, the rate constants correlate with the previously determined membrane fusogenicities. In membranes, exchange is retarded and a significant proportion of amide hydrogens are protected from exchange. We conclude that the conformational dynamics of SNARE TMD helices is mechanistically linked to their ability to drive lipid mixing.

[BIOPHYSICAL LETTERS] Are Current Molecular Dynamics Force Fields too Helical?

Best, R. B, Buchete, N.-V., Hummer, G. — 2008-05-02

Accurate force fields are essential for the success of molecular dynamics simulations. In apparent contrast to the conformational preferences of most force fields, recent NMR experiments (Graf et al., J. Am. Chem. Soc. 2007, 129, 1179-1189) suggest that short poly-alanine peptides in water populate the polyproline II structure almost exclusively. To investigate this apparent contradiction, with its ramifications for the assessment of molecular force fields and the structure of unfolded proteins, we have performed extensive simulations of Ala₅ in water (~5 µs total time), using twelve different force fields and three different peptide terminal groups. Using either empirical or density-functional based Karplus relations for the J-couplings, we find that most current force fields do overpopulate the region, with quantitative results depending on the Karplus relation and on the peptide termini. Even after re-weighting to match experiment, we find that Ala₅ retains significant and β populations. In fact, several force fields match the experimental data well before reweighting, and have a significant helical population. We conclude that radical changes to the best current force fields are not necessary, based on the NMR data. Nevertheless, the experiments of Graf et al. open the way toward the systematic improvement of current simulation models, such that they quantitatively reproduce the conformational equilibria of peptides.

[PROTEINS] The Fe2+ site of photosynthetic reaction centers probed by multiple scattering XAFS spectroscopy: improving structure resolution in dry matrices

2008-05-02

We report on the X-ray absorption fine structure (XAFS) of the Fe²⁺ site in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides. Crystallographic studies show that Fe²⁺ is ligated with four N atoms from four His residues and two O atoms from a Glu residue. By considering multiple scattering contributions to the XAFS function we improved the structural resolution of the site: His residues were split in two groups, characterized by different Fe-N distances, and two distinct Fe-O bond lengths resolved. The effect of the environment was studied by embedding the RC into a polyvinyl alcohol (PVA) film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-N distances, and in one Fe-O bond length, as compared to the PVA film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the RC surface and the water-trehalose matrix, which propagates deeply to the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster.

[ELECTROPHYSIOLOGY] Dynamics of the Preprotein Translocation Channel of the Outer Membrane of Mitochondria

Poynor, M. A., Eckert, R., Nussberger, S. — 2008-05-02

The protein translocase of the outer mitochondrial membrane TOM serves as the main entry site for virtually all mitochondrial proteins. Like many other protein translocases it also has an ion channel activity which can be used to study the dynamical properties of this supramolecular complex. We have purified TOM core complex and Tom40, the main pore forming subunit, from mitochondria of the filamentous fungus N. crassa and incorporated them into planar lipid bilayers. We then examined their single channel properties to provide a detailed description of the conformational dynamics of this channel in the absence of its protein substrate. For isolated TOM core complex we have found at least six conductance states. Transitions between these states were voltage-dependent with a bell-shaped open probability distribution and distinct kinetics depending on the polarity of the applied voltage. The states with the largest conductance followed an Ohmic I-V characteristic consistent with a large cylindrical pore with very little interaction with the permeating ions. For the lower conductance states, however, we have observed inverted S-shaped non-linear current-voltage curves reminiscent to those of much narrower pores where the permeating ions have to surmount an electrostatic energy barrier. At low voltages (less than ±70 mV), purified Tom40 protein did not show any transitions between its conductance states. Prolonged exposure to higher voltages induced similar gating behavior to what we observed for TOM core complex. This effect was time-dependent and reversible, indicating that Tom40 forms not only the pore but also contains the 'gating machinery' of the complex. However, for proper functioning, additional proteins (Tom22, Tom7, Tom6 and Tom5) are required that act as a modulator of the pore dynamics by significantly reducing the energy barrier between different conformational states.

[MEMBRANES] Interaction of Lipopolysaccharide and Phospholipid in Mixed Membranes: Solid-State 31P-NMR Spectroscopic and Microscopic Investigations

Nomura, K., Inaba, T., Morigaki, K., Brandenburg, K., Seydel, U., Kusumoto, S. — 2008-05-02

Lipopolysaccharide (LPS), which constitutes the outermost layer of Gram-negative bacterial cells as a typical component essential for their life, induces the first line defense system of innate immunity of higher animals. To understand the basic mode of interaction between bacterial LPS and phospholipid cell membranes, distribution patterns were studied by various physical methods of deep rough mutant LPS (ReLPS) of Escherichia coli incorporated in phospholipid bilayers as simple models of cell membranes. Solid-state ³¹P-NMR spectroscopic analysis suggested that a substantial part of ReLPS is incorporated into 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers when multilamellar vesicles (MLVs) were prepared from mixtures of these. In egg L--phosphatidylcholine (egg-PC)-rich membranes, ReLPS undergoes micellization. In phosphatidylethanolamine (PE)-rich membranes, however, micellization was not observed. We studied by microscopic techniques the location of ReLPS in membranes of ReLPS / egg-PC (1:10 M/M) and ReLPS / egg-PC / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (1:9:1 M/M/M). The influence of ReLPS on the physicochemical properties of the membranes was also studied as well. Microscopic images of both giant unilamellar vesicles (GUVs) and supported planar lipid bilayers (SPBs) showed that LPS was uniformly incorporated in the egg-PC lipid bilayers. In the egg-PC / POPG (9:1 M/M) lipid bilayers, however, ReLPS is only partially incorporated and becomes a part of the membrane in a form of aggregates (or as mixed aggregates with the lipids) on the bilayer surface. The lipid lateral diffusion coefficient measurements at various molar ratios of ReLPS / egg-PC / POPG indicated that the incorporated ReLPS reduces the diffusion coefficients of the phospholipids in the membrane. The retardation of diffusion became more significant with increasing POPG concentrations in the membrane at high ReLPS / phospholipid ratios. The present work demonstrated that the phospholipid composition has critical influence on the distribution of added ReLPS in the respective lipid membranes and also on the morphology and physicochemical property of the resulting membranes. A putative major factor causing these phenomena is reasoned to be the miscibility between ReLPS and individual phospholipid compositions.

[PROTEINS] sNASP, a histone H1-specific eukaryotic chaperone dimer that facilitates chromatin assembly

Finn, R. M., Browne, K., Hodgson, K. C., Ausio, J. — 2008-05-02

Nuclear autoantigenic sperm protein (NASP) has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but it is widely distributed throughout eukaryotes both in its somatic (sNASP) and testicular (tNASP) forms. The secondary structure of the human somatic version consists mainly of clusters of -helices and exists as a homodimer in solution. The protein binds non-specifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1.The formation of the NASP-H1 complexes is mediated by the N- and C-terminal domains of histone H1 and it does not involve the winged-helix domain which is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.

[BIOPHYSICAL THEORY AND MODELING] Are DNA Transcription Factor Proteins Maxwellian Demons?

Hu, L., Grosberg, A. Y., Bruinsma, R. — 2008-05-02

Transcription Factor (TF) proteins rapidly locate unique target sites on long genomic DNA molecules - and bind to them - during gene regulation. The search mechanism is known to involve a combination of 3D diffusion through the bulk of the cell and 1D sliding diffusion along the DNA. It is believed that the surprisingly high target binding rates of TF proteins relies on conformational fluctuations of the protein between a mobile state that is insensitive to the DNA sequence and an immobile state that is sequence sensitive. Since TF are not able to consume free energy during their search to obtain DNA sequence information, the Second Law of Thermodynamics must impose a strict limit on the efficiency of passive search mechanisms. In this paper we use a simple model for the protein conformational fluctuations to obtain the shortest binding time consistent with thermodynamics. The binding time is minimized if the spectrum of conformational fluctuations that take place during the search is "impedance-matched" to the large-scale conformational change that takes place at the target site. For parameter values appropriate for bacterial TF, this minimum binding time is within an order of magnitude of a limiting binding time corresponding to an idealized protein with instant target recognition. Numerical estimates suggest that typical bacteria operate in this regime of optimized conformational fluctuations.

[BIOPHYSICAL THEORY AND MODELING] Conduction of Na+ and K+ through the NaK Channel: Molecular and Brownian Dynamics Studies

Vora, T., Bisset, D., Chung, S.-H. — 2008-05-02

Conduction of ions through the NaK channel, with M0 helix removed, was studied using both Brownian dynamics and molecular dynamics. Brownian dynamics simulations predict that the truncated NaK has about a third of the conductance of the related KcsA K⁺ channel, is outwardly rectifying, and has a Michaelis-Menten current-concentration relationship. Current magnitude increases when the glutamine residue located near the intracellular gate is replaced with a glutamate residue. The channel is blocked by extracellular Ca²⁺. Molecular dynamics simulations show that, under the influence of a strong applied potential, both Na⁺ and K⁺ move across the selectivity filter, although conduction rates for Na⁺ ions are somewhat lower. The mechanism of conduction of Na+ differs significantly from that of K⁺ in that Na⁺ is preferentially coordinated by single planes of pore-lining carbonyl oxygens, instead of two planes as in the usual K⁺ binding sites. The water-containing `filter pocket' resulting from a single change in the selectivity filter sequence (compared to potassium channels) disrupts several of the planes of carbonyl oxygens, and thus reduces the filter's ability to discriminate against sodium.

[PROTEINS] BIOPHYSICAL CHARACTERIZATION OF THE UNSTRUCTURED CYTOPLASMIC DOMAIN OF THE HUMAN NEURONAL ADHESION PROTEIN NEUROLIGIN 3

2008-05-02

Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In the present study, we over-express and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the E. coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle X-ray scattering, circular dichroism, electron spin resonance, and NMR we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.

[PROTEINS] Defining the epitope region of a peptide from the Streptomyces coelicolor phosphoenolpyruvate: sugar phosphotransferase system able to bind to the enzyme I

2008-05-02

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the histidine-phosphocarrier protein, HPr, by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr^9-30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His15) and the first -helix of HPr of Streptomyces coelicolor, HPr^sc. By using fluorescence and circular dichroism, we firstly determined qualitatively that HPr^sc and HPr^9-30 did bind to EI^sc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr^9-30 and HPr^sc for EI^sc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr^9-30 was formed by residues Leu14, His15, Ile21 and Val23. The binding reaction between EI^sc and HPr^sc is enthalpy-driven and in other species is entropy-driven; further, the affinity of HPr^sc for EI^sc was smaller than in other species. However, the affinity of HPr^9-30 for EI^sc was only moderately lower than that of EI^sc for HPr^sc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.

[BIOPHYSICAL THEORY AND MODELING] Parameter Inference for Biochemical Systems that undergo a Hopf Bifurcation

Kirk, P. D.W., Toni, T., Stumpf, M. P.H. — 2008-05-02

The increasingly widespread use of parametric mathematical models to describe biological systems means that the ability to infer model parameters is of great importance. In this study, we consider parameter inferability in nonlinear ordinary differential equation models that undergo a bifurcation, focusing on a simple but generic biochemical reaction model. We systematically investigate the shape of the likelihood function for the model's parameters, analyzing the changes that occur as the model undergoes a Hopf bifurcation. We demonstrate that there exists an intrinsic link between inference and the parameters' impact on the modeled system's dynamical stability, which we hope will motivate further research in this area.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Diffusion of flexible random-coil dextran polymers measured in anisotropic brain extracellular space by integrative optical imaging

Xiao, F., Nicholson, C., Hrabe, J., Hrabetova, S. — 2008-05-02

There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging (IOI) method to measure diffusion of the fluorophore Alexa Fluor 488 (AF, MW 0.547 kDa) and fluorophore-labeled flexible random-coil dextran polymers (dexMW; MW 3, 75, 282, 525 kDa) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional (2-D) images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D^*_major and D^*_minor, decreased with molecular size. The diffusion anisotropy ratio (DAR = D^*_major/D^*_minor) increased for AF through dex75 but then, unexpectedly, reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform in order to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at about 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

[CELL BIOPHYSICS] The Visocelasticity of Membrane Tethers and its Importance for Cell Adhesion

Schmitz, J., Martin, B., Gottschalk, K. E. — 2008-05-02

Cell adhesion mechanically couples cells to surfaces. The durability of individual bonds between the adhesive receptors and their ligands in the presence of forces determines the cellular adhesion strength. For adhesive receptors like integrins, it is a common paradigm that the cell regulates its adhesion strength by altering the affinity state of the receptors. However, the probability distribution of rupture forces is not only dependent on the affinity of individual receptor-ligand bonds, but also on the mechanical compliance of the cellular anchorage of the receptor. Hence, by altering the anchorage, the cell can regulate its adhesion strength without changing the affinity of the receptor. Here, we analyze the anchorage of the integrin VLA-4 with its ligand VCAM-1. For this purpose, we develop a model based on the Kelvin body, which allows one to quantify the mechanical properties of the adhesive receptor's anchorage using atomic force microscopy on living cells. As we demonstrate, the measured force curves give valuable insight into the mechanics of the cellular anchorage of the receptor, which is described by the tether-stiffness, the membrane rigidity and the membrane viscosity. The measurements relate to a tether stiffness of k_t=1.6µN/m, an initial membrane rigidity of k_i=260µN/m and a viscosity of µ=5.9µN·s/m. Integrins exist in different activation states. When activating the integrin with Mg²⁺, we observe altered viscoelastic parameters of k_t=0.9µN/m, k_i=190µN/m and µ=6.0µN·s/m. Based on our model, we postulate that anchorage-related effects are common regulating mechanisms for cellular adhesion beyond affinity regulation.

[BIOPHYSICAL THEORY AND MODELING] Monte Carlo study of single molecule diffusion can elucidate the mechanism of B cell synapse formation

Tsourkas, P. K, Longo, M. L, Raychaudhuri, S. — 2008-05-02

B cell receptors have been shown to cluster at the intercellular junction between a B cell and an antigen-presenting cell (APC) in the form of a segregated pattern of B cell receptor/antigen (BCR/Ag) complexes known as an immunological synapse. We use a simple random walk based theoretical model and Monte Carlo simulations to study the effect of diffusion of surface-bound molecules on B cell synapse formation. Our results show that B cell synapse formation is optimal for a limited range of receptor-ligand complex diffusion coefficient values, typically one-to-two orders of magnitude lower than the diffusion coefficient of free receptors. Such lower mobility of receptor-ligand complexes can significantly affect the diffusion of a tagged receptor or ligand in an affinity dependent manner, as the binding/unbinding of such receptor or ligand molecules crucially depends on affinity. Our work shows how single molecule tracking experiments can be used to estimate the order of magnitude of the diffusion coefficient of receptor-ligand complexes, which is difficult to measure directly in experiments due to the finite life-time of receptor-ligand bonds. We also show how such antigen movement data at the single molecule level can provide insight into the B cell synapse formation mechanism. Thus, our results can guide further single molecule tracking experiments to elucidate the synapse formation mechanism in B cells, and potentially in other immune cells.

[BIOPHYSICAL THEORY AND MODELING] Molecular dynamics simulations of insertion of chemically modified DNA nanostructures into water-chloroform interface

Lin, J., Seeman, N. C, Vaidehi, N. — 2008-05-02

DNA based 2D and 3D arrays have been used as templates for synthesis of functional polymers and proteins. Hydrophobic or amphiphilic DNA arrays would be useful for the synthesis of hydrophobic molecules. The objective of this study is to design modified amphiphilic double crossover DX-DNA molecule that would insert into water-chloroform interface thus showing amphiphilic character. Since experiments for such design are tedious, we have used molecular dynamics simulations to identify and optimize the functional groups to modify the DNA backbone, that would enable insertion into the water-chloroform interface, prior to synthesis. By methylating the phosphates of the backbone, to make phosphonates, combined with placing a benzyl group at the 2' position of the deoxyribose rings in the backbone, we observed that the simple B-DNA structure was able to insert into the water-chloroform interface. We find that the transfer free energy of the methylated benzylated DNA is better than either just methylated or benzylated DNA. The driving force for this insertion comes from entropic contribution to the free energy and the favorable van der Waals interaction of the chloroform molecules with the methyl and benzyl groups of the DNA.

[BIOPHYSICAL LETTERS] The Molecular Density of States in Bacterial Nanowires

El-Naggar, M. Y, Gorby, Y. A, Xia, W., Nealson, K. H — 2008-04-25

The recent discovery of electrically conductive bacterial appendages has significant physiological, ecological, and biotechnological implications, but the mechanism of electron transport in these nanostructures remains unclear. We here report quantitative measurements of transport across bacterial nanowires produced by the dissimilatory metal-reducing bac-terium (DMRB), Shewanella oneidensis MR-1, whose electron transport system is being investigated for renewable energy recovery in microbial fuel cells and bioremediation of heavy metals and radionuclides. The Shewanella nanowires display a surprising non-linear electrical transport behavior, where the voltage dependence of the conductance reveals peaks indicat-ing discrete energy levels with higher electronic density of states. Our results indicate that the molecular constituents along the Shewanella nanowires possess an intricate electronic structure that plays a role in mediating transport.

[BIOPHYSICAL LETTERS] Starting structure dependence of NMR order parameters derived from MD simulations: Implications for judging force field quality

Koller, A. N., Schwalbe, H., Gohlke, H. — 2008-04-25

Comparing experimental generalized N-H S² order parameters to those calculated from MD trajectories is increasingly used to judge force field quality and completeness of sampling. Herein we demonstrate for the well investigated system hen egg white lysozyme that different experimental starting structures can lead to significant differences in MD-derived S² parameters that can be even larger than S² parameter deviations due to different force fields. Caution should thus be taken in general when simulated S² parameters are compared to experimental data with the aim of judging force field quality. We show that adequately sampling flexible regions (on the order of 100 ns) and only calculating S² parameters averaged over short time windows proofed necessary to obtain consistent results irrespective of the starting structure.

[PROTEINS] Flow Induced Structural Transition in the {beta}-switch Region of Glycoprotein Ib

Chen, Z., Lou, J., Zhu, C., Schulten, K. — 2008-04-25

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this paper we present a molecular dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue β-switch region of platelet glycoprotein Ib, for which crystal structures of its Nterminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a β-hairpin. Interaction between glycoprotein Ib and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to β-hairpin transition in glycoprotein Ib is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop -> β-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side group packing step.

[BIOPHYSICAL THEORY AND MODELING] The twilight zone between protein order and disorder

Szilagyi, A., Gyorffy, D., Zavodszky, P. — 2008-04-25

The amino acid composition of intrinsically disordered proteins and protein segments characteristically differs from that of ordered proteins. This observation forms the basis of several disorder prediction methods. These, however, usually perform worse for smaller proteins (or segments) than for larger ones. We show that the regions of amino acid composition space corresponding to ordered and disordered proteins overlap with each other, and the extent of the overlap (the "twilight zone") is larger for short than for long chains. To explain this finding, we used two-dimensional lattice model proteins containing hydrophobic, polar, and charged monomers, and revealed the relationship between chain length, amino acid composition, and disorder. Because the number of chain configurations exponentially grows with chain length, a larger fraction of longer chains can reach a low-energy, ordered state than shorter chains. The amount of information carried by the amino acid composition about whether a protein or segment is (dis)ordered grows with increasing chain length. Smaller proteins rely more on specific interactions for stability, which limits the possible accuracy of disorder prediction methods. For proteins in the "twilight zone", size can determine order, as illustrated by the example of two-state homodimers.

[MINI-REVIEW] Determination of protein structures - a series of fortunate events

Chruszcz, M., Wlodawer, A., Minor, W. — 2008-04-25

Determination of a macromolecular structure using X-ray diffraction is a multi-step process that involves a plethora of techniques involving molecular biology, bioinformatics, and physical sciences. Counterintuitively, the success of any or all individual steps does not guarantee the success of the overall process. This review examines the difficulties presented by each step on the path from a gene to the final publication, together with certain lucky (or unlucky) circumstances that can affect the velocity along that path.

[MINI-REVIEW] Cooperativity and Specificity in Enzyme Kinetics: A Single-Molecule Time-based Perspective

Qian, H. — 2008-04-25

An alternative theoretical approach to enzyme kinetics that is particularly applicable to single molecule enzymology is presented. The theory, originated by Van Slyke and Cullen in 1914, develops enzyme kinetics from a "time perspective" rather than the traditional "rate perspective", and emphasizes the nonequilibrium steady state nature of enzymatic reactions and the significance of small copy numbers of enzyme molecules in living cells. Sigmoidal cooperative substrate binding to slowly fluctuating, monomeric enzymes is shown to arise from association pathways with very small probability but extremely long passage time, which would be disregarded in the traditional rate perspective: A single enzyme stochastically takes alternative pathways in serial order rather than different pathways in parallel. The theory unifies dynamic cooperativity and Hopfield-Ninio's kinetic proofreading mechanism for specificity amplification.

[PROTEINS] Molten Globule and Native State Ensemble of Helicobacter pylori Flavodoxin. Can crowding, osmolytes or cofactors stabilize the native conformation relative to the molten globule?

Cremades, N., Sancho, J. — 2008-04-25

Partly unfolded protein conformations close in energy to the native state may be involved in protein functioning and also be related to folding diseases, but yet their structure and energetics are poorly understood. One such conformation, the monomeric and well-behaved molten globule of Helicobacter pylori apoflavodoxin, is here investigated to provide, in a wide pH interval, a complete thermodynamic description of its unfolding equilibrium and the equilibrium linking molten globule and native state. All thermodynamic and molecular properties of the molten globule here analyzed are characteristic of a partly unfolded conformation, and their differences with those of the native state are typically quantitative rather than qualitative. The stability data depict a native state ensemble where the relative populations of the different intermediates are strongly modulated by pH. While at pH 2.0 the molten globule is dominant, at neutral pH it is just the least stable of three partly unfolded intermediates populated by this protein. Interestingly, the energy rank of these intermediates at pH 7.0 is consistent with their likelihood to overcome the native state and become the more stable conformation when the native state protein is subjected to heat or mutation stress. Given the small volume difference between molten globule and native state, neither crowding agents nor osmolytes can drive the molten globule back to the native state. This observation, which is in qualitative accord with predictions of simple excluded volume theory, indicates that molecular crowding in vivo is not an effective mechanism to minimize partial unfolding events leading to equilibrium intermediates.

[BIOPHYSICAL THEORY AND MODELING] Diffusive coupling and network periodicity: a computational study

PARK, E.-H., FENG, Z., DURAND, D. M. — 2008-04-25

Diffusive coupling (nearest-neighbor coupling) is the most common type of coupling present in many systems (1-3). Previous experimental and theoretical studies have shown that potassium lateral diffusion coupling (i.e., diffusive coupling) can be responsible for synchronization of neuronal activity. Recent in-vivo experiments performed with anesthetized rat hippocampus suggested that the extracellular potassium could play an important role in the generation of a novel type of epileptiform non-synaptic activity. Yet, the role of potassium in the generation of seizures remains controversial. We tested the hypothesis that potassium lateral diffusion coupling is responsible for the coupling mechanisms for network periodicity in a nonsynaptic model of epilepsy in-vivo using a CA1 pyramidal neuron network model. The simulation results show that 1) potassium lateral diffusion coupling is crucial for establishing epileptiform activity similar to that generated experimentally; 2) there exists a scaling relation between the critical coupling strength and the number of cells in the network. The results not only agree with the theoretical prediction, but strongly suggest that potassium lateral diffusion coupling - a physiological realization of the concept of diffusive coupling - can play an important role in entraining periodicity in a non-synaptic neural network.