Biophysical Journal BioFAST

[PROTEINS] Electron-Electron Distances in Spin-Labeled Low-Spin Metmyoglobin Variants by Relaxation Enhancement

Ulyanov, D., Bowler, B. E, Eaton, G. R, Eaton, S. S — 2008-09-05

Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure gave protein with less hemichrome impurities than was obtained by overexpression of holo-protein followed by spin labeling. Coordination of cyanide to the met heme formed low-spin complexes. Iron-nitroxyl interspin distances in the range of 17 to 30 Å were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between about 30 and 140 K and by spin echo measurements of the enhancement of spin-spin relaxation rates at 10 to 30 K. Interspin distances also were calculated with the molecular modeling program Insight II®. For most of the variants, distances determined from the temperature dependence of spin echo intensities at a pulse spacing of 200 ns agree with distances measured by saturation recovery and calculated with Insight II® within about an Angstrom, which is within experimental uncertainties. Measurements of interspin distances via spin-spin relaxation enhancement have the advantages that maximum effects are observed for slower metal relaxation rates than are required for spin-lattice relaxation enhancement and the impact diminishes as r-3 instead of r-6 as for spin-lattice relaxation enhancement, which permits measurements at longer distances.

[PROTEINS] The Effect of Temperature on Mechanical Resistance of the Native and Intermediate States of I27

Taniguchi, Y., Brockwell, D. J, Kawakami, M. — 2008-09-05

We have investigated the effect of temperature on the mechanical unfolding of I27 from human cardiac titin, employing a custom-built temperature control device for single-molecule atomic force microscopy (AFM) measurement. A sawtooth pattern was observed in the force curves where each force peak reports on the unfolding of an I27 domain. In early unfolding events a 'hump-like' deviation was observed due to the detachment of β-strand A from each I27 domain. The force at which the humps appear was approximately 130 pN and showed no temperature dependence at least in the temperature range of 2 – 30 °C. The hump structure was successfully analysed with a two-state worm-like chain model and the Gibbs free energy difference of the detachment reaction was estimated to be 11.6 ± 0.58 kcal/mol and found to be temperature independent. By contrast, upon lowering the temperature, the mean unfolding force from the partly-unfolded intermediate state was found to markedly increase and the unfolding force distribution to broaden significantly, suggesting the distance (xu) between the folded and transition states in the energy landscape along the pulling direction is decreased. These results suggest that the local structure of β-strand A are stabilised by topologically-simple local hydrogen-bond network and the temperature does not affect the detachment reaction thermodynamically and kinetically, while the interaction between the β-strands A' and G, which is critical region for its mechanical stability, is strongly dependent on the temperature.

[SUPRAMOLECULAR ASSEMBLIES] Non-exponential kinetics of DNA escape from {alpha}-hemolysin nanopores

Wiggin, M., Tropini, C., Tabard-Cossa, V., Jetha, N. N., Marziali, A. — 2008-09-05

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from -hemolysin nanopores and show that anomalously long escape times for some events result in non-exponential kinetics. From the distribution of first passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

[PROTEINS] Hydration dynamics in a partially denatured ensemble of the globular protein human {alpha}-lactalbumin investigated with molecular dynamics simulations

Sengupta, N., Jaud, S., Tobias, D. J. — 2008-09-05

Atomistic molecular dynamics simulations are used to probe changes in the nature and the sub-nanosecond dynamical behavior of solvation waters that accompany partial denaturation of the globular protein, human -Lactalbumin (HLA). A simulated ensemble of sub-compact conformers, similar to the molten globule state of HLA, demonstrates a marginal increase in the amount of surface solvation relative to the native state. This increase is accompanied by subtle but distinct enhancement in surface water dynamics, less favorable protein-water interactions, and a marginal decrease in the anomalous behavior of solvation water dynamics. The extent of solvent influx is not proportional to the increased surface area and the partially denatured conformers are less uniformly solvated compared to their native counterpart. The observed solvation in partially denatured conformers is lesser in extent when compared to earlier experimental estimates in molten globule states, and consistent with more recent descriptions based on nuclear magnetic relaxation dispersion studies.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Contact Guidance Mediated 3D Cell Migration is Regulated by Rho/ROCK-dependent Matrix Reorganization

Provenzano, P. P, Inman, D. R, Eliceiri, K. W, Trier, S. M, Keely, P. J — 2008-09-05

Cells generate mechanical force to organize the extracellular matrix (ECM) and drive important developmental and reparative processes. Likewise, tumor cells invading into three-dimensional (3D) matrices remodel the ECM microenvironment. Importantly, we previously reported a distinct radial reorganization of the collagen matrix surrounding tumors that facilitates local invasion. Here we describe a mechanism by which cells utilize contractility events to reorganize the ECM to provide contact guidance that facilitates 3D migration. Using novel assays to differentially organize the collagen matrix we show that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of both human and mouse mammary epithelial cells. In contrast, organizing the collagen matrix to mimic the ECM organization associated with non-invading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/ROCK pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are pre-aligned matrices, inhibiting neither Rho nor ROCK inhibits 3D migration, supporting our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.

[MUSCLE AND CONTRACTILITY] Secondary Structure and Compliance of a Predicted Flexible Domain in Kinesin-1 Necessary for Co-operation of Motors

Crevenna, A. H, Madathil, S., Cohen, D. N, Wagenbach, M., Fahmy, K., Howard, J. — 2008-09-05

While the mechanism by which a kinesin-1 molecule moves individually along a microtubule is quite well understood, the way that many kinesin-1 motor proteins bound to the same cargo move together along a microtubule is not. We have identified a 60-aa-long domain, termed Hinge 1, in kinesin-1 from Drosophila melanogaster that is located between the coiled coils of the neck and stalk domains. Its deletion reduces microtubule gliding speed in multiple-motor assays but not single-motor assays. Hinge 1 thus facilitates the co-operation of motors by preventing them from impeding each other. We have addressed the structural basis for this phenomenon. Video-microscopy of single microtubule-bound full-length motors reveals the sporadic occurrence of high compliance states alternating with longer-lived low compliance states. Deletion of Hinge 1 abolishes transitions to the high compliance state. Based on FTIR, CD, and fluorescence spectroscopy of Hinge 1 peptides, we propose that the low compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas the high-compliance state corresponds to the loss of that structure. We hypothesize that strain accumulated during multiple-kinesin motility populates the high compliance state by unfolding helical secondary structure in the central Hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple-motor situations.

[PROTEINS] {alpha}-helical Topology Prediction and Generation of Distance Restraints in Membrane Proteins

McAllister, S. R, Floudas, C. A — 2008-09-05

The field of protein structure prediction has seen significant advances in recent years. Researchers have followed a multitude of approaches, including methods based on comparative modeling, fold recognition and threading, and first principles techniques. It is noteworthy that the structure prediction of membrane proteins is comparatively less studied by researchers in the field. A membrane protein is characterized by a protein structure that extends into or through the lipid-lipid bilayer of a cell. The structure is influenced by the combination of the hydrophobic bilayer region, the direct interaction with the bilayer, and the aqueous external environment. Due to the difficulty in obtaining reliable experimental structures, accurate computational prediction of membrane proteins is of paramount importance. An optimization model has been developed to predict the interhelical interactions in -helical membrane proteins. A database of -helical membrane proteins of known structure and limited sequence identity can be constructed to develop interaction probabilities. By then maximizing the occurrence of highly probable pairwise or three-residue interactions, realistic contacts can be predicted by imposing a number of geometrical constraints. The development of these low distance contacts can provide additional distance restraints for first principles-based approaches to the tertiary structure prediction problem. The proposed approach is shown to successfully predict interhelical contacts in several membrane protein systems, including bovine rhodopsin and the recently released human β2 adrenergic receptor protein structure.

[CELL BIOPHYSICS] Cell-cell mechanical communication through compliant substrates

King, C., Dembo, M., Hammer, D. A. — 2008-09-05

The role of matrix mechanics on cell behavior is under intense investigation. Cells exert contractile forces on their matrix and the matrix elasticity can alter these forces and cell migratory behavior. However, little is known about the contribution of matrix mechanics and cell-generated forces to stable cell-cell contact and tissue formation. Using matrices of varying stiffness and measurements of endothelial cell migration and traction stresses, we find that cells can detect and respond to substrate strains created by the traction stresses of a neighboring cell, and that this response is dependent on matrix stiffness. Specifically, pairs of endothelial cells (EC) display hindered migration on gels with elasticity below 5500 Pa in comparison to individual cells, suggesting these cells sense each other through the matrix. These results reveal for the first time that matrix mechanics can foster tissue formation by altering the relative motion between cells, promoting the formation of cell-cell contacts. Moreover, our data indicates that cells have the ability to communicate mechanically through their matrix. These findings are critical for the understanding of cell-cell adhesion during tissue formation and disease progression, and for the design of biomaterials intended to support both cell-matrix and cell-cell adhesion.

[BIOPHYSICAL THEORY AND MODELING] A Systematic Methodology for Defining Coarse-grained Sites in Large Biomolecules

Zhang, Z., Lu, L., Noid, W. G., Krishna, V., Pfaendtner, J., Voth, G. A. — 2008-08-29

Coarse-grained (CG) models of biomolecules have recently attracted considerable interest because they enable the simulation of complex biological systems on length-scales and time-scales that are inaccessible for atomistic molecular dynamics (MD) simulation. A CG model is defined by a map that transforms an atomically detailed configuration into a CG configuration. For CG models of relatively small biomolecules or in cases that the CG and all-atom models have similar resolution, the construction of this map is relatively straightforward and can be guided by chemical intuition. However, it is more challenging to construct a CG map when large and complex domains of biomolecules have to be represented by relatively few CG sites. The present work introduces a new and systematic methodology called essential dynamics coarse-graining (ED-CG). This approach constructs a CG map of the primary sequence at a chosen resolution for an arbitrarily complex biomolecule. In particular, the resulting ED-CG method variationally determines the CG sites that reflect the essential dynamics characterized by principal component analysis (PCA) of an atomistic MD trajectory. Numerical calculations illustrate this approach for the HIV-1 CA protein dimer and ATP-bound G-actin. Importantly, since the CG sites are constructed from the primary sequence of the biomolecule, the resulting ED-CG model may be better suited to appropriately explore protein conformational space than those from other CG methods at the same degree of resolution.

[BIOPHYSICAL LETTERS] Multiplexed FRET to image multiple signaling events in live cells

2008-08-29

We report a novel approach for simultaneous imaging of two different FRET sensors in the same cell with minimal spectral cross-talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross-talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximise the spectral separation between our chosen fluorophores whilst at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells following EGF stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.

[CELL BIOPHYSICS] Low Spring Constant Regulates P-selectin-PSGL-1 Bond Rupture

Zhang, Y., Sun, G., Lu, S., Li, N., Long, M. — 2008-08-29

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, as well as tumor metastasis. While the bond rupture has been well known at high loading rate r_f (≥10² pN/s), defined as the product of spring constant k and retract velocity v, the mechanism how the low r_f (<10² pN/s) or the low k regulates the bond dissociation, however, remains unclear. Here an optical trap (OT) assay was used to quantify the bond rupture at r_f ≤ 20 pN/s with low k (~10^-3-10^-2 pN/nm) when P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) were respectively coupled onto two glass microbeads. Our data indicated that the bond rupture force f retained the similar values when r_f increased up to 20 pN/s. It was also found that f varied with different combinations of k and v even at same r_f. Most probable force, f^*, was enhanced with spring constant when k < 47.0x10^-3 pN/nm, indicating that the bond dissociation at low r_f was spring constant-dependent and that bond rupture force depended on both loading rate and mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin-PSGL-1 bond dissociation at low r_f or k.

[BIOPHYSICAL THEORY AND MODELING] Influence of macromolecular crowding on protein-protein association rates - a Brownian Dynamics study

Wieczorek, G. A., Zielenkiewicz, P. — 2008-08-29

The high total concentration of macromolecules, often referred to as macromolecular crowding, is one of the characteristic features of living cells. Macromolecular crowding influences interactions between many types of macromolecules, with consequent effects on, among others, the rates of reactions occurring in the cell. Simulations to study the influence of crowding on macromolecular association rate were performed using a modified Brownian Dynamics protocol. The calculated values of the time-dependent self-diffusion coeffcients in different crowding conditions are in a very good agreement with those obtained by other authors. Simulations of the complex formation between the monoclonal antibody HyHEL-5 and its antigen hen egg lysosyme, both represented at atomic level detail, show that the calculated condensation rates strongly depend on the volume excluded by crowding. The rate obtained for the highest concentration of crowding particles is more than twofold higher than the rate for proteins without crowding.

[BIOPHYSICAL THEORY AND MODELING] All-Atom Computer Simulations of Amyloid Fibrils Disaggregation

Wang, J., Tan, C., Chen, H.-F., Luo, R. — 2008-08-29

Amyloid-like fibrils are found in many fatal diseases, including Alzheimer's disease, type II diabetes mellitus, transmissible spongiform encephalopathies, and prion diseases. These diseases are linked to proteins that have partially unfolded, misfolded, and aggregated into amyloid-like fibrils. The kinetics of amyloid-like fibrils aggregation is still hotly debated and remains an important open question. We have utilized the GNNQQNY crystal structure and high-temperature molecular dynamics simulation in explicit solvent to study the disaggregation mechanism of the GNNQQNY fibrils and to infer its likely aggregation pathways. A hexamer model and a 12mer model both with two parallel β sheets separated by a dry side-chain interface were adopted in our computational analysis. A cumulative time of 1 µs was simulated for the hexamer model at five different temperatures (298K, 348K, 398K, 448K, and 498K), and a cumulative time of 2.1 µs was simulated for the 12mer model at four temperatures (298K, 398K, 448K, and 498K). Our disaggregation landscape and kinetics analyses indicate that tetramers probably act as the transition state in both the hexamer and the 12mer simulations. In addition the 12mer simulations show that the initial aggregation nucleus is with 8 peptides. Furthermore the landscape is rather flat from 8mers to 12mers, indicating the absence of major barriers once the initial aggregation nucleus forms. Thus the likely aggregation pathway is from monomers to the initial nucleus of 8mers with tetramers as the transition state. Transition state structure analysis shows that the two dominant transition state conformations are tetramers in the 3-1 and 2-2 arrangements. The predominant nucleus conformations are in peptide arrangements maximizing dry side-chain contacts. Landscape and kinetics analyses also indicate that the parallel β sheets form earlier than the dry side-chain contacts during aggregation. These results provide further insights in understanding the early fibrils aggregation.

[BIOPHYSICAL LETTERS] High-speed high-resolution imaging of intercellular immune synapses using optical tweezers

2008-08-22

Imaging in any plane other than horizontal in a microscope typically requires a reconstruction from multiple optical slices that significantly decreases the spatial and temporal resolution that can be achieved. This can limit the precision with which molecular events can be detected, for example, at intercellular contacts. This has been a major issue for the imaging of immune synapses between live cells, which has generally required the reconstruction of en face intercellular synapses, yielding spatial resolution significantly above the diffraction limit and updating at only a few frames per minute. Strategies to address this issue have usually involved using artificial activating substrates such as antibody-coated slides or supported planar lipid bilayers, but synapses with these surrogate stimuli may not wholly resemble immune synapses between two cells. Here, we combine optical tweezers and confocal microscopy to realize generally applicable, high-speed, high-resolution imaging of almost any arbitrary plane of interest. Applied to imaging immune synapses in live-cell conjugates, this has enabled the characterization of complex behavior of highly-dynamic clusters of T cell receptors at the T cell / antigen-presenting cell intercellular immune synapse and revealed the presence of numerous, highly-dynamic long receptor-rich filopodial structures within inhibitory Natural Killer cell immune synapses.

[BIOENERGETICS] Functional halt positions of rotary FOF1-ATPase correlated with crystal structures

Sielaff, H., Rennekamp, H., Engelbrecht, S., Junge, W. — 2008-08-22

F_OF₁-ATPase is a rotary molecular motor. Driven by ATP-hydrolysis its central shaft rotates in 80° and 40° steps, interrupted by catalytic and ATP-waiting dwells. Rotation and halts were recorded by micro-videography in laboratory coordinates. A correlation with molecular coordinates was established by using an engineered pair of cysteines which, under oxidizing conditions, formed a zero-length cross-links between the rotor and the stator in an orientation as found in crystals. The fixed orientation coincided with the one of the catalytic dwell, whereas the ATP waiting dwell was displaced from it by +40°. In crystals the convex side of the cranked central shaft faces an empty nucleotide binding site, as if holding it open for arriving ATP. Functional studies have suggested three occupied sites during the catalytic dwell. Our data imply that the convex side rather faces a nucleotide-occupied than an empty site. The enzyme conformation in crystals thus seems to differ from the one during either dwell of the active enzyme. A revision of current schemes of the mechanism is proposed.

[PROTEINS] AN ALTERED MODE OF CALCIUM COORDINATION IN METHIONINE-OXIDIZED CALMODULIN

Jones, E. M., Squier, T. C., Sacksteder, C. A. — 2008-08-22

Oxidation of methionine residues in calmodulin (CaM) lowers the affinity for calcium and results in an inability to fully activate target proteins. To evaluate the structural consequences of CaM oxidation, we have used infrared difference spectroscopy to identify oxidation-dependent effects on the protein conformation and calcium liganding. Oxidation-induced changes include an increase in the hydration of -helices as indicated in the downshift of the amide I^' band of both apo- and Ca²⁺- CaM, and a modification of calcium liganding by carboxylate side chains reflected in antisymmetric carboxylate band shifts. Changes in carboxylate ligands are consistent with a model in which we propose: an Asp at position 1 of the EF-loop experiences diminished hydrogen bonding with the polypeptide backbone, an Asp at position 3 forms a bidentate coordination of calcium, and an Asp at position 5 forms a pseudobridging coordination with a calcium-bound water molecule. The bidentate coordination of calcium by conserved glutamates is unaffected by oxidation. The observed changes in calcium ligation are discussed in terms of the placement of methionine side chains relative to the calcium binding sites, which suggests that varying sensitivities of the binding sites to oxidation may underlie the loss of CaM function upon oxidation.

[SUPRAMOLECULAR ASSEMBLIES] Efficiency of histidine-associating compounds for blocking the Alzheimer's A{beta} channel activity and cytotoxicity

Arispe, N. J, Diaz, J. C, Flora, M. — 2008-08-22

The opening of the Alzheimer's Aβ channel permits the flux of calcium into the cell, thus critically disturbing intracellular ion homeostasis. Peptide segments which include the characteristic histidine diad, His₁₃ and His₁₄, efficiently block the Aβ channel activity blocking Aβ cytotoxicity. We hypothesize that the vicinal His-His peptides coordinate with the rings of His in the mouth of the pore, thus blocking the flow of calcium ions through the channel, with consequent blocking of Aβ cytotoxicity. To test this hypothesis we have studied Aβ ion channel activity and cytotoxicity, after the addition of compounds that are known to have histidine association capacity, such as Ni²⁺, imidazole, histidine and a series of histidine-related compounds. All compounds were effective at blocking both Aβ channels and preventing Aβ cytotoxicity. The efficiency of protection of histidine-related compounds was correlated with the number of imidazole side chains in the blocker compounds. These data reinforce the premise that histidine residues within the Aβ channel sequence are in the pathway of ion flow. Additionally, the data confirm the contribution of the Aβ channel to the cytotoxicity of exogenous Aβ.

[BIOPHYSICAL LETTERS] Coevolution of Function and the Folding Landscape: Correlation with Density of Native Contacts

Hills, Jr., R. D, Brooks III, C. L. — 2008-08-15

The relationship between the folding landscape and function of evolved proteins is explored by comparison of the folding mechanisms for members of the flavodoxin fold. CheY, Spo0F and NtrC have unrelated functions and low sequence homology but share an identical topology. Recent coarse-grained simulations show that their folding landscapes are uniquely tuned to properly suit their respective biological functions. Enhanced packing in Spo0F and its limited conformational dynamics compared to CheY or NtrC lead to frustration in its folding landscape. Simulation as well as experimental results correlate with the local density of native contacts for these and a sample of other proteins. In particular, protein regions of low contact density are observed to become structured late in folding; concomitantly, these dynamic regions are often involved in binding or conformational rearrangements of functional importance. These observations help to explain the widespread success of Go-like coarse-grained models in reproducing protein dynamics.

[BIOPHYSICAL LETTERS] FCS Study of Thermodynamics of Membrane Protein Insertion into Lipid Bilayer Chaperoned by Fluorinated Surfactants

Posokhov, Y. O, Rodnin, M. V, Das, S. K, Pucci, B., Ladokhin, A. S. — 2008-08-15

Experimental determination of the free energy (G) stabilizing the structure of membrane proteins (MPs) in their native environment has been hampered by MP's aggregation and precipitation outside the lipid bilayer. Recently we have demonstrated that the latter process can be prevented by the use of fluorinated surfactants, FTACs, which act as chaperones for MP insertion without partitioning in the membrane themselves (Rodnin et al., 2008 Biophys.J, 94:4348-4357). Here we combine the advantages of the chaperone-like ability of FTACs with the sensitivity of fluorescence correlation spectroscopy measurements to determine G of bilayer insertion of model MPs. First, we calibrate our approach by examining the effects of chaperoned insertion on G of transmembrane insertion of Annexin B12. We find that a shorter-chained surfactant, FTAC-C6, for which the working concentration range of 0.05-0.2 mM falls below CMC=0.33 mM, has a mild effect on an apparent G. In contrast, additions of a longer-chained FTAC-C8 (CMC=0.03 mM) result in a steep and non-linear concentration-dependence of G. Then, we applied the same methodology to the pH-triggered insertion of diphtheria toxin T-domain, which is known to be affected by non-productive aggregation in solution. We find that the correction of the G value needed to compensate for un-chaperoned insertion of the T-domain exceeds 3 kcal/mole. A relatively shallow and linear dependence of the G for Annexin B12 and T-domain insertion on FTAC-C6 concentration is encouraging for future applications of this surfactant in thermodynamic studies of the stability of other MPs.

[BIOPHYSICAL LETTERS] Nucleotide capacitance calculation for DNA sequencing

Lu, J.-Q., Zhang, X. — 2008-08-15

Using a first-principles linear response theory, the capacitance of the DNA nucleotides, adenine, cytosine, guanine and thymine, are calculated. The difference in the capacitance between the nucleotides is studied with respect to conformational distortion. The result suggests that although an alternate current capacitance measurement of a single-stranded DNA chain threaded through a nano-gap electrodes may not be sufficient to be used as a stand alone method for rapid DNA sequencing, the capacitance of the nucleotides should be taken into consideration in any GHz-frequency electric measurements and may also serve as an additional criterion for identifying the DNA sequence.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] DNA Translocation Governed by Interactions with Solid State Nanopores

Wanunu, M., Sutin, J., McNally, B., Chow, A., Meller, A. — 2008-08-15

We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the range 3 - 5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules in the range 150 - 3500 bp we find an exponent of 1.40, whereas for longer molecules an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of blocked ion current by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, while resulting in a more complex dynamics than previously observed with larger pores. Our results shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

[PROTEINS] Neither Helix in the Coiled Coil Region of the Axle of F1-ATPase Plays a Significant Role in Torque Production

2008-08-15

F₁-ATPase is an ATP-driven rotary molecular motor in which the central subunit rotates inside the cylinder made of ₃β₃ subunits. The amino and carboxy termini of the subunit form the axle, an -helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at same levels, resulting in slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25 to 40 amino acid residues. Longer truncation impaired the stability of the motor complex severely, 40 deletion failing to yield rotating one. Up to 36 deletion, however, the mutants produced an apparent torque nearly half the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low, due to load-dependent stumbling at 120° intervals even with saturating ATP. Comparison with the previous work indicates that half the normal torque is produced at the orifice of the stator, the very tip of the carboxy terminus adds the other half, while neither helix in the middle of the axle contributes little to torque generation and rapid progress of catalysis. None of the residues of the entire axle plays a specific decisive role in rotation.

[BIOPHYSICAL THEORY AND MODELING] Molecular Mechanism of Ion-Ion and Ion-Substrate Coupling in the Na+ -dependent leucine transporter LeuT

Caplan, D. A., Subbotina, J. O., Noskov, S. Y. — 2008-08-15

Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) plays an important role in the regulation of neuronal signaling. One of the ma jor events in the trans- port cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high- resolution structure for a bacterial homologue of Na+ / Cl- -dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only about 6 Å away. Extensive, fully atomistic, MD and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate binding affinity as a function of the ion load (single vs double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na+ and not to Li+ or K+ cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as "amplified" selectivity for Na+ in the case of double ion occupancy.

[PHOTOBIOPHYSICS] Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy

2008-08-15

BLUF (Blue Light sensing Using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino-acid side chains, including a highly conserved tyrosine and glutamine. Here, the participation of particular amino-acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD^•- and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, demonstrating that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay upon H/D exchange. Subsequent protonation of FAD^•- to result in FADH^• on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH^• C=N stretch marker mode, with tyrosine as the likely proton donor. FADH^• is reoxidized in 67 ps (180 ps in D₂O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch reveals infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by ~180° through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.

[MEMBRANES] The Activity of the Amphipathic Peptide {delta}-Lysin Correlates with Phospholipid Acyl Chain Structure and Bilayer Elastic Properties

Pokorny, A., Kilelee, E. M, Wu, D., Almeida, P. F — 2008-08-15

Release of lipid vesicle content induced by the amphipathic peptide -lysin was investigated as a function of lipid acyl chain length and degree of unsaturation for a series of phosphatidylcholines. Dye efflux and peptide binding were examined for three homologous lipid series: di-monounsaturated, di-polyunsaturated, and asymmetric phosphatidylcholines, with one saturated and one monounsaturated acyl chain. Except for the third series, peptide activity correlated with the first moment of the lateral pressure profile, which is a function of lipid acyl chain structure. In vesicles composed of asymmetric phosphatidylcholines, peptide binding and dye efflux are enhanced compared to symmetric, unsaturated lipids with similar pressure profiles. We attribute this to the entropically more favorable interaction of -lysin with partially saturated phospholipids. We find that lipid acyl chain structure has a major impact on the activity of -lysin and is likely to be an important factor contributing to the target specificity of amphipathic peptides.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] A DEND mutation in Kir6.2 (KCNJ11) reveals a flexible N-terminal region critical for ATP-sensing of the KATP channel

Koster, J. C., Kurata, H. T., Enkvetchakul, D., Nichols, C. G. — 2008-08-15

ATP-sensitive K⁺-channels link metabolism and excitability in neurons, myocytes and pancreatic islets. Mutations in the pore-forming subunit (Kir6.2; KCNJ11) cause neonatal diabetes, developmental delay and epilepsy, by decreasing sensitivity to ATP inhibition and suppressing electrical activity. Mutations of residue G53 underlie both mild (G53R,S) and severe (G53D) forms of the disease. All examined substitutions (G53D,R,S,A,C,F) reduce ATP-sensitivity, indicating intolerance for any amino acid other than glycine. Surprisingly, each mutation reduces ATP affinity, rather than intrinsic gating, even though structural modeling places G53 at a significant distance from the ATP-binding pocket. We propose that glycine is required in this location for flexibility of the distal N-terminus, and induced fit of ATP at the binding site. Consistent with this hypothesis, glycine substitution of the adjacent residue (Q52G) partially rescues ATP affinity of reconstituted Q52G/G53D channels. The results reveal an important feature of the non-canonical ATP sensing mechanism of K_ATP channels.

[MEMBRANES] Simulations of skin barrier function: Free energies of hydrophobic and hydrophilic transmembrane pores in ceramide bilayers

Notman, R., Anwar, J., Briels, W J, Noro, M. G, den Otter, W. K — 2008-08-15

Transmembrane pore formation is central to many biological processes such as ion transport, cell fusion and viral infection. Furthermore, pore formation in the ceramide bilayers of the stratum corneum may be an important mechanism by which penetration enhancers such as dimethylsulfoxide (DMSO) weaken the barrier function of the skin. We have used the potential of mean constraint force (PMCF) method to calculate the free energy of pore formation in ceramide bilayers in both the innate gel phase and in the DMSO-induced fluidized state. Our simulations show that the fluid phase bilayers form archetypal water-filled hydrophilic pores similar to those observed in phospholipid bilayers. In contrast, the rigid gel-phase bilayers develop hydrophobic pores. At the relatively small pore diameters studied here, the hydrophobic pores are empty rather than filled with bulk water, suggesting that they do not compromise the barrier function of ceramide membranes. A phenomenological analysis suggests that these vapor pores are stable, below a critical radius, because the penalty of creating water-vapor and tailvapor interfaces is lower than that of directly exposing the strongly hydrophobic tails to water. The PMCF free energy profile of the vapor pore supports this analysis. The simulations indicate that high DMSO concentrations drastically impair the barrier function of the skin by strongly reducing the free energy required for pore opening.

[CELL BIOPHYSICS] Hemoglobin Dynamics in Red Blood Cells: Correlation to Body Temperature

Stadler, A. M., Digel, I., Artmann, G., Embs, J. P., Zaccai, G., Buldt, G. — 2008-08-15

A transition in hemoglobin behavior close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBC) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperature of a variety of species. In an exploration of the molecular basis of the transition, we present neutron scattering measurements on the temperature dependence of hemoglobin dynamics in whole human RBC, in vivo. The data revealed a change in the geometry of internal protein motions at 36.9 °C, at human body temperature. Above that temperature, amino acid side-chain motions occupy bigger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBC was also measured and compared favorably with theoretical predictions for short-time self-diffusion of non-charged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and Ångstrom length scale might well be connected to a macroscopic effect on whole RBC occurring at body temperature.

[BIOPHYSICAL THEORY AND MODELING] Multiple subunit fitting into a low-resolution density map of a macromolecular complex using Gaussian mixture model

Kawabata, T. — 2008-08-15

Recently, electron microscopy measurement of single-particles has enabled us to reconstruct a low-resolution 3D density map of large bio-molecular complexes. If structures of the complex subunits can be solved by X-ray crystallography at atomic resolution, fitting these models into the 3D density map can generate an atomic resolution model of the entire large complex. The fitting of multiple subunits, however, generally requires large computational costs; therefore, a development of an efficient algorithm is required. We developed a new fast fitting program, "gmfit", which employs a Gaussian mixture model to represent approximated shapes of the 3D density map and the atomic models. A Gaussian mixture model is a distribution function composed by the sum of several 3D Gaussian density functions. Because our model analytically provides an integral of a product of two distribution functions, it enables us to quickly calculate the fitness of the density map and the atomic models. Using the integral, two types of potential energy functions are introduced: the attraction potential energy between a 3D density map and each subunit, and the repulsion potential energy between subunits. The restraint energy for symmetry is also employed to build symmetrical origomeric complexes. To find the optimal configuration of subunits, we randomly generated initial configurations of atomic models, and performed a steepest descent method using forces and torques of the two potential energies. Comparison between an original density map and its Gaussian mixture model showed that the required number of Gaussian distribution functions for a given accuracy depended on both resolution and molecular size. We then performed test fitting calculations for simulated low-resolution density maps of atomic models of homo dimer, trimer and hexamer, using different search parameters. The results indicated that our method was able to rebuild atomic models of complex even for 30 Å resolution maps, if sufficient numbers (>=8) of Gaussian distribution functions were employed for each subunit, and the symmetric restraints were assigned for complexes with more than three subunits. As a more realistic test, we tried to build an atomic model of the GroEL/ES complex by fitting 21 subunit atomic models into the 3D density map obtained by cryo-EM using the C7 symmetric restraints. A low-RMSD (14.7 Å) model was obtained as the lowest energy model, showing that our fitting method was reasonably accurate. Inclusion of other restraints from biological and biochemical experiments could further enhance the accuracy.

[MEMBRANES] Phenomenological Model and Phase Behavior of Saturated and Unsaturated Lipids and Cholesterol

Putzel, G. G., Schick, M. — 2008-08-15

We present a phenomenological theory for the phase behavior of ternary mixtures of cholesterol and saturated and unsaturated lipids, one which describes both liquid and gel phases. It leads to the following description of the mechanism of the phase behavior. In a binary system of the lipids, phase separation occurs when the saturated chains are well ordered, as in the gel phase, simply due to packing effects. In the liquid phase the saturated ones are not sufficiently well ordered for separation to occur. The addition of cholesterol, however, increases the saturated lipid order to the point that phase separation is once again favorable. Our theory addresses this last mechanism, the means by which cholesterol-mediated ordering of membrane lipids leads to liquid-liquid immiscibility. It produces, for the system above the main chain transition of the saturated lipid, phase diagrams in which there can be liquid-liquid phase separation in the ternary system but not in any of the binary ones, while below that temperature it yields the more common phase diagram in which a gel phase, rich in saturated lipid, appears in addition to the two liquid phases.

[BIOPHYSICAL THEORY AND MODELING] Growing actin networks form lamellipodium and lamellum by self-assembly

Huber, F., Kas, J., Stuhrmann, B. — 2008-08-15

Many different cell types are able to migrate by formation of a thin actin based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures designated lamellipodium and lamellum which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic 2D computer simulation that includes chemical reaction kinetics, g-actin diffusion and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In the present work we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1-2 µm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin or tropomyosin dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data is compared to existing experimental data.

[BIOPHYSICAL THEORY AND MODELING] New Proposed Mechanism of Actin-Polymerization-Driven Motility

Lee, K.-C., Liu, A. J. — 2008-08-15

We present the first numerical simulation of actin-driven propulsion by elastic filaments. Specifically, we use a Brownian dynamics formulation of the dendritic nucleation model of actin-driven propulsion. We show that the model leads to a self-assembled network that exerts forces on a disk and pushes it with an average speed. This simulation approach is the first to observe a speed that varies non-monotonically with the concentration of branching proteins (Arp2/3), capping protein and depolymerization rate (ADF), in accord with experimental observations. Our results suggest a new interpretation of the origin of motility. When we estimate the speed that this mechanism would produce in a system with realistic rate constants and concentrations as well as fluid flow, we obtain a value that is within an order of magnitude of the polymerization speed deduced from experiments.

[BIOPHYSICAL THEORY AND MODELING] Models of Toxic {beta}-sheet Channels of Protegrin-1 (PG-1) Suggest a Common Subunit Organization Motif Shared with Toxic Alzheimer {beta}-Amyloid Ion Channels

Jang, H., Ma, B., Lal, R., Nussinov, R. — 2008-08-15

Antimicrobial peptides (AMPs) induce cytotoxicity by altering membrane permeability. The electrical properties of membrane-associated AMPs as well as their cellular effects have been extensively documented; however their 3D structure is poorly understood. Gaining insight into channel structures is important to the understanding of the PG-1 and other AMPs' cytolytic mechanisms and to antibiotics design. Here we studied the β-sheet channels morphology using molecular dynamics (MD) simulations. We modeled protegrin-1 (PG-1) channels as intrinsic barrel-stave and toroidal membrane pores, and simulated them in zwitterionic and anionic lipid bilayers. PG-1 channels consist of eight β-hairpins in a consecutive NCCN (N and C represent the β-hairpin's N- and C-termini) packing organization yielding antiparallel and parallel β-sheet channels. Both channels preserve the toroidal, but not the barrel-stave pores. The two lipid leaflets of the bilayer bend toward each other at the channels' edges, producing a 'semi'-toroidal pore with the outward-pointing hydrophobic residues preventing the polar lipid head-groups from moving to the bilayer center. In all simulated lipid environments, PG-1 channels divide into 4 or 5 β-sheet subunits consisting of single or dimeric β-hairpins. The channel morphology with subunit organization is consistent with the 4 to 5 subunits observed by NMR in the POPE/POPG bilayer. Remarkably, a β-sheet subunit channel motif is in agreement with Alzheimer ion channels modeled using the universal U-shape β-strand-turn-β-strand structure, as well as with high resolution AFM images of β-amyloid channels with 4-6 subunits. Consistent with the toxic β-amyloid channels which are ion-conducting, the PG-1 channels permeate anions.

[PROTEINS] Structure of SP-B/DPPC Mixed Films Studied by Neutron Reflectometry

Fullagar, W. K, Holt, S. A, Gentle, I. R — 2008-08-15

The structures of films of SP-B and mixtures of SP-B and DPPC at the air/water interface have been studied by neutron reflectometry (NR) and Langmuir film balance methods. From the film balance studies we observe that at very high pressures the isotherms of pure DPPC and SP-B/DPPC mixtures very nearly overlay one another, suggesting that the SP-B is being excluded from the film. The use of multiple contrasts with NR at a range of surface pressures has enabled the mixing and 'squeeze out' of the DPPC and SP-B mixtures to be studied. We are able to identify the SP-B component of the interfacial structure and its position as a function of surface pressure. The mixtures are initially a homogeneous layer at low surface pressures. At higher surface pressures the SP-B is squeezed out of the lipid layer into the subphase, with the first signs detected at 30 mN m^-1. At 50 mN m^-1 the subphase is almost completely excluded from the DPPC layer with the SP-B content significantly reduced. Only a small amount of DPPC appears to be associated with the squeezed out SP-B.

[CELL BIOPHYSICS] Measurement of 2D binding constants between cell bound MHC and immobilized antibodies with an acoustic biosensor

Saitakis, M., Dellaporta, A., Gizeli, E. — 2008-08-15

Gaining insight into the dynamic processes of the molecular interactions that mediate cell-substrate and cell-cell adhesion is of great significance to the understanding of the numerous physiological processes driven by intercellular communication. In this work an acoustic wave biosensor is employed to study and characterize the specific interaction between cell-bound membrane proteins to surface immobilized ligands using as a model system the binding of the MHC class I HLA-A2 proteins to anti-HLA-A2 monoclonal antibodies. The energy of the acoustic signal, measured as amplitude change, was found to depend directly on the number of HLA-A2/antibody complexes formed on the device surface. Real-time acoustic data was used to monitor the surface binding of cell suspensions in the range of 6.0x10⁴ to 6.0x10⁵ cells ml^-1. Membrane interactions are governed by two-dimensional (2D) chemistry due to the molecules' confinement to the lipid bilayer; the 2D kinetics and affinity constant of the HLA-A2/antibody interaction were calculated (k_a = 1.15x10^-5 µm² s^-1 per molecule, k_d = 2.07x10^-5 s^-1 and K_A = 0.556 µm² per molecule, at 25°C) based on a detailed acoustic data analysis. Results indicate that acoustic biosensors can emerge as a significant tool for probing and characterizing cell membrane interactions in the immune system as well as for the fast and label-free screening of membrane molecules using whole cells.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Long lasting synchronization of calcium oscillations by cholinergic stimulation in isolated pancreatic islets

2008-08-15

Individual mouse pancreatic islets exhibit oscillations in [Ca²⁺]i and insulin secretion in response to glucose in vitro but how the oscillations of a million islets are coordinated within the human pancreas in vivo is unclear. Islet to islet synchronization is necessary, however, for the pancreas to produce regular pulses of insulin. To determine whether neurohormone release within the pancreas might play a role in coordinating islet activity, [Ca²⁺]i changes in 4-6 isolated mouse islets were simultaneously monitored before and after a transient pulse of a putative synchronizing agent. The degree of synchronicity was quantified using a novel analytical approach that yields a parameter that we call the "Synchronization Index" (SI). Individual islets exhibited [Ca²⁺]i oscillations with periods of 3-6 minutes, but were not synchronized under control conditions. However, raising islet [Ca²⁺]i with a brief application of the cholinergic agonist carbachol (25 µM) or elevated KCl in glucose-containing saline rapidly synchronized islet [Ca²⁺]i oscillations for > 30 minutes, long after the synchronizing agent was removed. In contrast, the adrenergic agonists clonidine or norepinephrine, and the K_ATP channel inhibitor tolbutamide failed to synchronize islets. Partial synchronization was observed, however, with the K_ATP channel opener diazoxide. The synchronizing action of carbachol depended on the glucose concentration used, suggesting that glucose metabolism was necessary for synchronization to occur. In order to understand how transiently perturbing islet [Ca²⁺]i produced sustained synchronization, we used a mathematical model of islet oscillations in which complex oscillatory behavior results from the interaction between a fast electrical subsystem and a slower metabolic oscillator. Transient synchronization simulated by the model was mediated by resetting of the islet oscillators to a similar initial phase followed by transient "ringing" behavior, during which the model islets oscillated with a similar frequency. These results suggest that neurohormone release from intra-pancreatic neurons could help synchronize islets in situ. Defects in this coordinating mechanism, could contribute to the disrupted insulin secretion observed in Type 2 diabetes.

[CELL BIOPHYSICS] Quantitative Measurement of cAMP Concentration Using an Epac Based FRET-Sensor

Salonikidis, P. S., Zeug, A., Kobe, F., Ponimaskin, E., Richter, D. W. — 2008-08-15

FRET-based biosensors for the quantitative analysis of intracellular signalling, including sensors for monitoring cyclic AMP are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration, but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores.
Here we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an "exchange protein directly activated by cAMP" (Epac). In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by cAMP changes was determined as E = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in range from 150 nM to 15 µM. The method described is also suitable for other FRET based biosensors with a 1:1 donor/acceptor stoichiometry.
As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels following stimulation of the Gs-coupled serotonin 5-HT7 receptor.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Anion-Cation Permeability Correlates with Hydrated Counter-ion Size in Glycine Receptor Channels

Sugiharto, S., Lewis, T. M., Moorhouse, A. J., Schofield, P. R., Barry, P. H. — 2008-08-15

The functional role of ligand-gated ion channels depends critically on whether they are predominantly permeable to cations or anions. However these, and other ion channels, are not perfectly selective, allowing some counter-ions to also permeate. To address the mechanisms by which such counter-ion permeation occurs, we measured the anion-cation permeabilities of different alkali cations, Li⁺, Na⁺, Cs⁺, relative to either Cl^-, or NO₃^- anions in both a WT glycine receptor channel (GlyR) and a mutant GlyR with a wider pore diameter. We hypothesized and showed that counter-ion permeation in anionic channels correlated inversely with an equivalent or effective hydrated size of the cation relative to the channel pore radius, with larger counter-ion permeabilities being observed in the wider pore channel. We also showed that the anion component of conductance was independent of the nature of the cation. We suggest that anions and counter-ion cations can permeate through the pore as neutral ion pairs, to allow the cations to overcome the large energy barriers resulting from the positively charged selectivity filter in small GlyR channels, with the permeability of such ion-pairs being dependent on the effective hydrated diameter of the ion pair relative to the pore diameter.

[BIOPHYSICAL THEORY AND MODELING] A steroid in a lipid bilayer: Localization, orientation and energetics

Vijayan, R., Biggin, P. C — 2008-08-08

Steroid hormones are known to freely partition into lipid bilayers. As a case study, we investigated the behavior of the steroid hormone cortisone in a model lipid bilayer. First, we looked at energy barriers involved in the partitioning of a single molecule into a bilayer using umbrella sampling molecular dynamics simulations. A rather wide well of -4.5 kcal/mol was observed in the interfacial region between the lipid headgroup and tailgroup. Next, using two unconstrained molecular dynamics simulation with cortisone initially positioned at distinct locations within a bilayer, we studied the preferred location and orientation of the molecule. Finally, we observed how cortisone molecules could spontaneously insert and localize in a bilayer from bulk solution. The three independent approaches produced a converged picture of how cortisone behaves in a model lipid bilayer.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Diffusional Channeling in the Sulfate Activating Complex: Combined Continuum Modeling and Coarse-grained Brownian Dynamics Studies

2008-08-08

Enzymes required for sulfur metabolism have been suggested to gain effciency by restricted diffusion ("channeling") of an intermediate APS^2- between active sites. This article describes modeling of the whole channeling process by numerical solution of the Smoluchowski diffusion equation, as well as by coarse-grained Brownian dynamics. The results suggest that electrostatics plays an essential role in the APS^2- channeling. Furthermore, with coarse-grained Brownian dynamics, the substrate channeling process has been studied with reactions in multiple active sites. Our simulations provide a bridge for numerical modeling with Brownian dynamics to simulate the complicated reaction and diffusion and raise important questions relating to the electrostatically mediated substrate channeling in vitro, in situ and in vivo.

[CELL BIOPHYSICS] Cooperativity in Adhesion Cluster Formation during Initial Cell Adhesion

Selhuber-Unkel, C., Lopez-Garcia, M., Kessler, H., Spatz, J. P. — 2008-08-08

We have studied the initial phase of cell adhesion as a function of the lateral organization of individual integrin molecules with single-cell force microscopy. Nanostructures, consisting of hexagonally ordered gold dots, were prepared with diblock-copolymer micelle lithography and functionalized with RGD peptides, thus defining integrin position with nanometer resolution. Adhesion strength was characterized with an atomic force microscope and both cell detachment forces and work of detachment revealed a reinforcement of adhesion if the distance between integrin molecules was less than 70 nm. This reinforcement occurred already at cell-substrate contact times less than five minutes. Our results demonstrate quantitatively the relevance of the distance between adjacent integrin binding sites rather than their density. Furthermore, we propose a model describing the cooperative stabilization of early integrin clusters as a function of receptor patterning at the nano-scale.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Site-specific Effects of Compression on Macromolecular Diffusion in Articular Cartilage

Leddy, H. A., Guilak, F. — 2008-08-08

Articular cartilage is the connective tissue that lines joints and provides a smooth surface for joint motion. Because cartilage is avascular, molecular transport occurs primarily via diffusion or convection, and cartilage matrix structure and composition may affect diffusive transport. Due to the inhomogeneous compressive properties of articular cartilage, we hypothesized that compression would decrease macromolecular diffusivity and increase diffusional anisotropy in a site-specific manner that depends on local tissue strain. We utilized two fluorescence photobleaching methods, SCAMP (Scanning Microphotolysis) and FICOPP (Fluorescence Imaging of Continuous Point Photobleaching), to measure diffusion coefficients and diffusional anisotropy of 70kDa dextran in cartilage during compression and measured local tissue strain using texture correlation. For every 10% increase in normal strain, fractional change in diffusivity decreased by 0.16 in all zones and diffusional anisotropy increased 1.1 fold in the surface, 1.04 fold in the middle, and did not change in the deep zone. These results indicate that inhomogeneities in matrix structure and composition may significantly affect local diffusive transport in cartilage, particularly in response to mechanical loading. Our findings suggest that the high strains in the surface zone significantly decrease diffusivity and increase anisotropy, which may decrease transport between cartilage and synovial fluid during compression.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy

Rinia, H., Burger, K. N. J., Bonn, M., Muller, M. — 2008-08-08

Lipid droplets (LDs) are highly dynamic organelles that carry out multiple functions including the regulated storage and release of cholesterol and fatty acids. Information on the molecular composition of individual LDs within their cellular context is crucial to understand the diverse biological functions of LDs as well as their involvement in the development of metabolic disorders such as obesity, type II diabetes, and atherosclerosis. Although ensembles of LDs isolated from cells and tissues have been analyzed in great detail, quantitative information on the heterogeneity in lipid composition of individual droplets and possible variations within single lipid droplets is lacking. Therefore, we developed a label-free quantitative method to image lipids within LDs in 3T3-L1 cells. The method combines sub-micron spatial resolution in three dimensions using label-free coherent anti-Stokes Raman scattering (CARS) microscopy with quantitative analysis based on the maximum entropy method. Our method allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual LDs. Our results reveal variations in lipid composition and physical state between LDs contained in the same cell, and even within a single LD.

[CELL BIOPHYSICS] Membrane Deformation Under Local pH Gradient: Mimicking Mitochondrial Cristae Dynamics

KHALIFAT, N., PUFF, N., BONNEAU, S., FOURNIER, J.-B., ANGELOVA, M. I. — 2008-08-08

Mitochondria are cell sub-structures (organelles) critical for cell life, because biological "fuel" production, the ATP synthesis by oxidative phosphorylation, occurs in them driven by acidity (pH) gradients. Mitochondria play a key role as well in the cell death and in various fatigue and exercise intolerance syndromes. It is clear now that mitochondria present an astonishing variety of inner membrane morphologies, dynamically correlated with their functional state, coupled with the rate of the ATP synthesis, and characteristic for normal as well as for pathological cases. Our work offers some original insights into the factors that determine the dynamical tubular structures of the inner membrane cristae. We show the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid membrane. We designed a minimal membrane system (giant unilamellar vesicles, GUV) and experimentally showed that the directional modulation of local pH gradient at membrane level of cardiolipin-containing vesicles induces dynamic cristae-like membrane invaginations. We propose a mechanism and theoretical model to explain the observed tubular membrane morphology and suggest the underlying role of cardiolipin. Our results support the hypothesis of localized bioenergetic transduction and contribute to revealing the inherent capacity of cristae morphology to become self-maintaining and to optimize the ATP synthesis.

[PROTEINS] Free-Energy Linkage between Folding and Calcium Binding in EF-Hand Proteins

Suarez, M. C., Rocha, C. B., Sorenson, M. M., Silva, J. L., Foguel, D. — 2008-08-08

Troponin is the singular Ca²⁺-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca²⁺-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties in spite of their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca²⁺ was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (V and G_atm). Herein, we propose that part of the affinity for Ca²⁺ is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca²⁺ binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca²⁺ in EF-hand proteins, which is crucial to function.

[BIOPHYSICAL THEORY AND MODELING] Structure and Interactions of Aggrecans: Statistical Thermodynamic Approach

Nap, R., Szleifer, I. — 2008-08-08

Weak polyelectrolytes tethered to cylindrical surfaces are investigated using a molecular theory. These polymers form a model system to describe the properties of aggrecan molecules, which is one of the main components of cartilage. We have studied the structural and thermodynamical properties of two interacting aggrecans with a molecular density functional theory that incorporates the acid-base equilibrium as well as the molecular properties: including conformations, size, shape, and charge distribution of all molecular species. The effect of acidity and salt concentration upon the behavior is explored in detail. The repulsive interactions between two cylindrical shaped aggrecans are strongly influenced by both the salt concentration and the pH. With increasing acidity the polyelectrolytes of the aggrecan acquire charge and with decreasing salt concentration those charges become less screened. Consequently the interactions increase in size and range with increasing acidity and decreasing salt concentration. The size and range of the forces offers a possible explanation to the aggregation behavior of aggrecans and for their ability to resist compressive forces in cartilage. Likewise, the interdigitation of two aggrecan molecules is strongly affected by the salt concentration as well as the pH. With increasing pH the amount of charges increases causing the repulsions between the polymers to increase, leading to a lower interdigitation of the two cylindrical polymer layers of the aggrecan molecules. The low interdigitation in charged polyelectrolytes layers provides an explanation for the good lubrication properties of polyelectrolytes layers in general and cartilage in particular.

[MEMBRANES] High-Vapor Pressure Perfluorocarbons Cause Vesicle Fusion and Changes in Membrane Packing

Venegas, B., Wolfson, M., Cooke, P., Chong, P. L. — 2008-08-08

Perfluorocarbons (PFCs) hold great promise for biomedical applications. However, relatively little is known regarding the impact of these chemicals on membranes. The present work uses unilamellar vesicles to explore the effects of PFCs on membrane packing and vesicle stability. Four clinically relevant PFCs with varying vapor pressures (PP1 (294 mbar); PP2 (141 mbar); PP4, (9.6 mbar); PP9 (2.9 mbar)) were examined. Microscopy imaging and spectroscopic measurements suggest that PFCs, especially those with high vapor pressures, lead to vesicle fusion within hours. Upon exposure to PP1 and PP2 for 72 hrs, vesicles retain a spherical shape, but the size is changed from ~200 nm to ~20-40 µm. Additionally, membrane packing undergoes marked changes during this time frame. A significant decrease in water content in the lipid polar headgroup regions occurs during the first 1-2 hours exposure to PFCs, followed by a steady increase in water content over time. Possible mechanisms have been proposed to explain these dramatic structural changes. The finding that the chemically inert PFCs exhibit a fusogenic activity and marked changes in membrane surface packing is novel and should be considered when using PFCs for biomedical applications.