Biophysical Journal current issue

[NEW AND NOTABLE] Dynamic Organization of Gene Loci and Transcription Compartments in the Cell Nucleus

Spudich, J. A. — 2008-11-18

[BIOPHYSICAL THEORY AND MODELING] Dynamics of Voltage Profile in Enzymatic Ion Transporters, Demonstrated in Electrokinetics of Proton Pumping Rhodopsin

Hagedorn, R., Gradmann, D., Hegemann, P. — 2008-11-18

H⁺-pumping rhodopsins mediate a primordial conversion of light to metabolic energy. Bacteriorhodopsin from Halobacterium salinarium is the first identified and (biochemically) best-studied H⁺-pumping rhodopsin. The electrical properties of H⁺-pumping rhodopsins, however, are known in more detail for the homolog Acetabularia rhodopsin, isolated from the eukaryotic green alga Acetabularia acetabulum. Based on data from Acetabularia rhodopsin we present a general reaction kinetic model of H⁺-pumping rhodopsins with only seven independent parameters, which fits the kinetic properties of photocurrents as functions of light, transmembrane voltage, internal and external pH, and time. The model describes fast photoisomerization of retinal with simultaneous H⁺ transfer to an H⁺ acceptor, reprotonation of retinal from the intracellular face via an H⁺ donor, and proton release to the extracellular space via an H⁺ release complex. The voltage sensitivities of the individual reaction steps and their temporal changes are treated here by a novel approach, whereby—as in an Ohmic voltage divider—the effective portions of the total transmembrane voltage decrease with the relative velocities of the individual reaction steps. This analysis quantitatively infers dynamic changes of the voltage profile and of the pK values of the H⁺-binding sites involved.

[BIOPHYSICAL THEORY AND MODELING] A Simplified Model of Local Structure in Aqueous Proline Amino Acid Revealed by First-Principles Molecular Dynamics Simulations

Troitzsch, R. Z., Tulip, P. R., Crain, J., Martyna, G. J. — 2008-11-18

Aqueous proline solutions are deceptively simple as they can take on complex roles such as protein chaperones, cryoprotectants, and hydrotropic agents in biological processes. Here, a molecular level picture of proline/water mixtures is developed. Car-Parrinello ab initio molecular dynamics (CPAIMD) simulations of aqueous proline amino acid at the B-LYP level of theory, performed using IBM's Blue Gene/L supercomputer and massively parallel software, reveal hydrogen-bonding propensities that are at odds with the predictions of the CHARMM22 empirical force field but are in better agreement with results of recent neutron diffraction experiments. In general, the CPAIMD (B-LYP) simulations predict a simplified structural model of proline/water mixtures consisting of fewer distinct local motifs. Comparisons of simulation results to experiment are made by direct evaluation of the neutron static structure factor S(Q) from CPAIMD (B-LYP) trajectories as well as to the results of the empirical potential structure refinement reverse Monte Carlo procedure applied to the neutron data.

[BIOPHYSICAL THEORY AND MODELING] Free-Energy Profiles of Membrane Insertion of the M2 Transmembrane Peptide from Influenza A Virus

Yeh, I.-C., Olson, M. A., Lee, M. S., Wallqvist, A. — 2008-11-18

The insertion of the M2 transmembrane peptide from influenza A virus into a membrane has been studied with molecular-dynamics simulations. This system is modeled by an atomically detailed peptide interacting with a continuum representation of a membrane bilayer in aqueous solution. We performed replica-exchange molecular-dynamics simulations with umbrella-sampling techniques to characterize the probability distribution and conformation preference of the peptide in the solution, at the membrane interface, and in the membrane. The minimum in the calculated free-energy surface of peptide insertion corresponds to a fully inserted, helical peptide spanning the membrane. The free-energy profile also shows that there is a significant barrier for the peptide to enter into this minimum in a nonhelical conformation. The sequence of the peptide is such that hydrophilic amino acid residues at the ends of the otherwise primarily hydrophobic peptide create a trapped, U-shaped conformation with the hydrophilic residues associated with the aqueous phase and the hydrophobic residues embedded in the membrane. Analysis of the free energy shows that the barrier to insertion is largely enthalpic in nature, whereas the membrane-spanning global minimum is favored by entropy.

[BIOPHYSICAL THEORY AND MODELING] Influence of Macromolecular Crowding on Protein-Protein Association Rates--a Brownian Dynamics Study

Wieczorek, G., Zielenkiewicz, P. — 2008-11-18

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 coefficients 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 lysozyme, both represented at atomic level detail, show that the calculated association rates strongly depend on the volume excluded by crowding. The rate obtained for the highest concentration of crowding particles is greater 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. — 2008-11-18

Amyloidlike 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 amyloidlike fibrils. The kinetics of amyloidlike 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 12-mer 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 (298 K, 348 K, 398 K, 448 K, and 498 K), and a cumulative time of 2.1 µs was simulated for the 12-mer model at four temperatures (298 K, 398 K, 448 K, and 498 K). Our disaggregation landscape and kinetics analyses indicate that tetramers probably act as the transition state in both the hexamer and the 12-mer simulations. In addition, the 12-mer simulations show that the initial aggregation nucleus is with eight peptides. Furthermore, the landscape is rather flat from 8-mers to 12-mers, 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 8-mers 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 THEORY AND MODELING] Gap Junction Coupling and Calcium Waves in the Pancreatic Islet

Benninger, R. K. P., Zhang, M., Head, W. S., Satin, L. S., Piston, D. W. — 2008-11-18

The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of β-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet.

[BIOPHYSICAL THEORY AND MODELING] The Selectivity of K+ Ion Channels: Testing the Hypotheses

Fowler, P. W., Tai, K., Sansom, M. S. P. — 2008-11-18

How K⁺ channels are able to conduct certain cations yet not others remains an important but unresolved question. The recent elucidation of the structure of NaK, an ion channel that conducts both Na⁺ and K⁺ ions, offers an opportunity to test the various hypotheses that have been put forward to explain the selectivity of K⁺ ion channels. We test the snug-fit, field-strength, and over-coordination hypotheses by comparing their predictions to the results of classical molecular dynamics simulations of the K⁺ selective channel KcsA and the less selective channel NaK embedded in lipid bilayers. Our results are incompatible with the so-called strong variant of the snug-fit hypothesis but are consistent with the over-coordination hypothesis and neither confirm nor refute the field-strength hypothesis. We also find that the ions and waters in the NaK selectivity filter unexpectedly move to a new conformation in seven K⁺ simulations: the two K⁺ ions rapidly move from site S4 to S2 and from the cavity to S4. At the same time, the selectivity filter narrows around sites S1 and S2 and the carbonyl oxygen atoms rotate 20°–40° inwards toward the ion. These motions diminish the large structural differences between the crystallographic structures of the selectivity filters of NaK and KcsA and appear to allow the binding of ions to S2 of NaK at physiological temperature.

[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. — 2008-11-18

Coarse-grained (CG) models of biomolecules have recently attracted considerable interest because they enable the simulation of complex biological systems on length-scales and timescales that are inaccessible for atomistic molecular dynamics 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. This 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 of an atomistic molecular dynamics 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 THEORY AND MODELING] Modeling by Assembly and Molecular Dynamics Simulations of the Low Cu2+ Occupancy Form of the Mammalian Prion Protein Octarepeat Region: Gaining Insight into Cu2+-Mediated {beta}-Cleavage

Pushie, M. J., Vogel, H. J. — 2008-11-18

The prion protein has garnered considerable interest because of its involvement in prion disease as well as its unresolved cellular function. The octarepeat region in the flexible N-domain is capable of binding copper through multiple coordination modes. Under conditions of low pH and low Cu²⁺ concentration, the four octarepeats (ORs) cooperatively coordinate a single copper ion. Based on the average structure of the PHGG and GWGQ portions of a copper-free OR₂ model from molecular dynamics simulations, the starting structures of the OR₄ complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu²⁺ coordination. Molecular dynamics simulations of Cu²⁺ bound to the assembled OR₄ model (Cu:OR₄) reveal a close association of specific Trp and Gly residues with the Cu²⁺ center. This low Cu²⁺-occupancy form of prion protein is redox-active and can readily initiate cleavage of the OR region, mediated by reactive oxygen species generated by Cu⁺. The OR region is known to be required for β-cleavage, as are the Trp residues within the OR region. The β-cleaved form of the prion protein accumulates in amyloid fibrils. Hence, the close approach of Trp and Gly residues to the Cu²⁺ coordination site in the low Cu²⁺-occupancy form of the OR region may signal an important interaction for the initiation of prion disease.

[BIOPHYSICAL THEORY AND MODELING] Light Transmission in the Human Cornea as a Function of Position across the Ocular Surface: Theoretical and Experimental Aspects

Doutch, J., Quantock, A. J., Smith, V. A., Meek, K. M. — 2008-11-18

This article investigates the theoretical basis for differences in visible light transmission through the human cornea as a function of distance from the center. Experimentally, transmission decreases approximately linearly up to 3 mm from the central axis, then quadratically beyond this. It is known that collagen fibril number density and collagen fibril radii change from the central region to the corneal periphery. We modeled, using the direct-summation-of-scattered-fields method, the effects these ultrastructural changes would be expected to have on light transmission, accounting for the increase in corneal thickness from center to edge. Fibril positions for the modeling were obtained from electron micrographs of human cornea. Theoretically, transmission remains fairly constant across the central cornea; then, as the fibril diameter increases, the predicted scattering increases. Interfibrillar spacing changes alter the refractive index ratio between matrix and fibril; this was modeled in our theoretical deductions. Fibril number density had a minimal effect on light propagation. Our theoretical deductions were in broad agreement with our experimental data. It is concluded that the reduced transparency in the peripheral stroma is primarily caused by changes in fibril radius and an increase in refractive index ratio between the fibril and the interfibrillar substance.

[BIOPHYSICAL THEORY AND MODELING] Dimer Opening of the Nucleotide Binding Domains of ABC Transporters after ATP Hydrolysis

Wen, P.-C., Tajkhorshid, E. — 2008-11-18

ABC transporters constitute one of the most abundant membrane transporter families. The most common feature shared in the family is the highly conserved nucleotide binding domains (NBDs) that drive the transport process through binding and hydrolysis of ATP. Molecular dynamics simulations are used to investigate the effect of ATP hydrolysis in the NBDs. Starting with the ATP-bound, closed dimer of MalK, four simulation systems with all possible combinations of ATP or ADP-P_i bound to the two nucleotide binding sites are constructed and simulated with equilibrium molecular dynamics for ~70 ns each. The results suggest that the closed form of the NBD dimer can only be maintained with two bound ATP molecules; in other words, hydrolysis of one ATP can lead to the opening of the dimer interface of the NBD dimer. Furthermore, we observed that the opening is an immediate effect of hydrolysis of ATP into ADP and P_i rather than the dissociation of hydrolysis products. In addition, the opening is mechanistically triggered by the dissociation of the LSGGQ motif from the bound nucleotide. A metastable ADP-P_i bound conformational state is consistently observed before the dimer opening in all the simulation systems.

[BIOPHYSICAL THEORY AND MODELING] A Spatial Toggle Switch Drives Boundary Formation in Development

Canela-Xandri, O., Sagues, F., Reigada, R., Buceta, J. — 2008-11-18

Herein we introduce a multicellular network motif that performs as a spatial toggle switch and explains how boundary formation can be faithfully accomplished in developmental processes. Importantly, we show that expression and activity patterns of proteins must be simultaneously characterized for a proper understanding and description of the underlying mechanism. Our in silico experiments, in agreement with in vivo results, evaluate different genetic backgrounds and shed light on the dynamics of boundary formation. In addition, we provide an estimation of relevant biological parameters and a robustness analysis.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Determinants within the Turret and Pore-Loop Domains of KCNQ3 K+ Channels Governing Functional Activity

Zaika, O., Hernandez, C. C., Bal, M., Tolstykh, G. P., Shapiro, M. S. — 2008-11-18

KCNQ1–5 (Kv7.1–7.5) subunits assemble to form a variety of functional K⁺ channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by ~56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA⁺-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba²⁺ block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position "unlocking" the channels into a conductive conformation.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Bupivacaine Blocks N-Type Inactivating Kv Channels in the Open State: No Allosteric Effect on Inactivation Kinetics

Nilsson, J., Madeja, M., Elinder, F., Arhem, P. — 2008-11-18

Local anesthetics bind to ion channels in a state-dependent manner. For noninactivating voltage-gated K channels the binding mainly occurs in the open state, while for voltage-gated inactivating Na channels it is assumed to occur mainly in inactivated states, leading to an allosterically caused increase in the inactivation probability, reflected in a negative shift of the steady-state inactivation curve, prolonged recovery from inactivation, and a frequency-dependent block. How local anesthetics bind to N-type inactivating K channels is less explored. In this study, we have compared bupivacaine effects on inactivating (Shaker and K_v3.4) and noninactivating (Shaker-IR and K_v3.2) channels, expressed in Xenopus oocytes. Bupivacaine was found to block these channels time-dependently without shifting the steady-state inactivation curve markedly, without a prolonged recovery from inactivation, and without a frequency-dependent block. An analysis, including computational testing of kinetic models, suggests binding to the channel mainly in the open state, with affinities close to those estimated for corresponding noninactivating channels (300 and 280 µM for Shaker and Shaker-IR, and 60 and 90 µM for K_v3.4 and K_v3.2). The similar magnitudes of K_d, as well as of blocking and unblocking rate constants for inactivating and noninactivating Shaker channels, most likely exclude allosteric interactions between the inactivation mechanism and the binding site. The relevance of these results for understanding the action of local anesthetics on Na channels is discussed.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Potential Cation and H+ Binding Sites in Acid Sensing Ion Channel-1

Shaikh, S. A., Tajkhorshid, E. — 2008-11-18

Acid sensing ion channels (ASICs) are cation-selective membrane channels activated by H⁺ binding upon decrease in extracellular pH. It is known that Ca²⁺ plays an important modulatory role in ASIC gating, competing with the ligand (H⁺) for its binding site(s). However, the H⁺ or Ca²⁺ binding sites involved in gating and the gating mechanism are not fully known. We carried out a computational study to investigate potential cation and H⁺ binding sites for ASIC1 via all-atom molecular dynamics simulations on five systems. The systems were designed to test the candidacy of some acid sensing residues proposed from experiment and to determine yet unknown ligand binding sites. The ion binding patterns reveal sites of cation (Na⁺ and Ca²⁺) localization where they may compete with protons and influence channel gating. The highest incidence of Ca²⁺ and Na⁺ binding is observed at a highly acidic pocket on the protein surface. Also, Na⁺ ions fill in an inner chamber that contains a ring of acidic residues and that is near the channel entrance; this site could possibly be a temporary reservoir involved in ion permeation. Some acidic residues were observed to orient and move significantly close together to bind Ca²⁺, indicating the structural consequences of Ca²⁺ release from these sites. Local structural changes in the protein due to cation binding or ligand binding (protonation) are examined at the binding sites and discussed. This study provides structural and dynamic details to test hypotheses for the role of Ca²⁺ and Na⁺ ions in the channel gating mechanism.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Impaired Ca2+-Dependent Activation of Large-Conductance Ca2+-Activated K+ Channels in the Coronary Artery Smooth Muscle Cells of Zucker Diabetic Fatty Rats

Lu, T., Ye, D., He, T., Wang, X.-l., Wang, H.-l., Lee, H.-C. — 2008-11-18

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] A Mutation in CFTR Modifies the Effects of the Adenylate Kinase Inhibitor Ap5A on Channel Gating

Dong, Q., Randak, C. O., Welsh, M. J. — 2008-11-18

Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P¹,P⁵-di(adenosine-5')pentaphosphate (Ap₅A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap₅A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap₅A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap₅A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap₅A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis.

[CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING] Mechanism of KCl Enhancement in Detection of Nonionic Polymers by Nanopore Sensors

Rodrigues, C. G., Machado, D. C., Chevtchenko, S. F., Krasilnikov, O. V. — 2008-11-18

The mechanisms of KCl-induced enhancement in identification of individual molecules of poly(ethylene glycol) using solitary -hemolysin nanoscale pores are described. The interaction of single molecules with the nanopore causes changes in the ionic current flowing through the pore. We show that the on-rate constant of the process is several hundred times larger and that the off-rate is several hundred times smaller in 4 M KCl than in 1 M KCl. These shifts dramatically improve detection and make single molecule identification feasible. KCl also changes the solubility of poly(ethylene glycol) by the same order of magnitude as it changes the rate constants. In addition, the polymer-nanopore interaction is determined to be a strong non-monotonic function of voltage, indicating that the flexible, nonionic poly(ethylene glycol) acts as a charged molecule. Therefore, salting-out and Coulombic interactions are responsible for the KCl-induced enhancement. These results will advance the development of devices with sensor elements based on single nanopores.

[MEMBRANES] Probing the Lipid Membrane Dipole Potential by Atomic Force Microscopy

Yang, Y., Mayer, K. M., Wickremasinghe, N. S., Hafner, J. H. — 2008-11-18

The electrostatic properties of biological membranes can be described by three parameters: the transmembrane potential, the membrane surface potential, and the membrane dipole potential. The first two are well characterized in terms of their magnitudes and biological effects. The dipole potential, however, is not well characterized. Various methods to measure the membrane dipole potential indirectly yield different values, and there is not even agreement on the source of the membrane dipole moment. This ambiguity impedes investigations into the biological effects of the membrane dipole moment, which should be substantial considering the large interfacial fields with which it is associated. Electrostatic analysis of phosphatidylcholine lipid membranes with the atomic force microscope reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipids. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internal membrane dipole potential. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported phosphatidylcholine membranes. This new ability to quantitatively measure the membrane dipole moment in a noninvasive manner with nanometer scale spatial resolution will be useful in identifying the biological effects of the dipole potential.

[MEMBRANES] The Gaussian Curvature Elastic Energy of Intermediates in Membrane Fusion

Siegel, D. P. — 2008-11-18

The Gaussian curvature elastic energy contribution to the energy of membrane fusion intermediates has usually been neglected because the Gaussian curvature elastic modulus, , was unknown. It is now possible to measure for phospholipids that form bicontinuous inverted cubic (Q_II) phases. Here, it is shown that one can estimate for lipids that do not form Q_II phases by studying the phase behavior of lipid mixtures. The method is used to estimate for several lipid compositions in excess water. The values of are used to compute the curvature elastic energies of stalks and catenoidal fusion pores according to recent models. The Gaussian curvature elastic contribution is positive and similar in magnitude to the bending energy contribution: it increases the total curvature energy of all the fusion intermediates by 100 units of k_BT or more. It is important to note that this contribution makes the predicted intermediate energies compatible with observed lipid phase behavior in excess water. An order-of-magnitude fusion rate equation is used to estimate whether the predicted stalk energies are consistent with the observed rates of stalk-mediated processes in pure lipid systems. The current theory predicts a stalk energy that is slightly too large, by ~30 k_BT, to rationalize the observed rates of stalk-mediated processes in phosphatidylethanolamine or N-monomethylated dioleoylphosphatidylethanolamine systems. Despite this discrepancy, the results show that models of fusion intermediate energy are accurate enough to make semiquantitative predictions about how proteins mediate biomembrane fusion. The same rate model shows that for proteins to drive biomembrane fusion at observed rates, they have to perform mediating functions corresponding to a reduction in the energy of a purely lipidic stalk by several tens of k_BT. By binding particular peptide sequences to the monolayer surface, proteins could lower fusion intermediate energies by altering the elastic constants of the patches of lipid monolayer that form the stalk. Here, it is shown that if peptide binding changes or some other combinations of local elastic constants by only tens of percents, the stalk energy and the energy of catenoidal fusion pores would decrease by tens of k_BT relative to the pure lipid value. This is comparable to the required mediating effect. The curvature energies of stalks and catenoidal fusion pores have almost the same dependence on monolayer elastic constants as the curvature energies of the rhombohedral and Q_II phases; respectively. The effects of isolated fusion-relevant peptides on the energies of these intermediates can be determined by studying the effects of the peptides on the stability of rhombohedral and Q_II phases.

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

2008-11-18

Although 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 identified a 60-amino-acid-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 cooperation of motors by preventing them from impeding each other. We 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. The deletion of Hinge 1 abolishes transitions to the high-compliance state. Based on Fourier transform infrared, circular dichroism, and fluorescence spectroscopy of Hinge 1 peptides, we propose that low-compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas high-compliance states correspond 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.

[MUSCLE AND CONTRACTILITY] Alternative Versions of the Myosin Relay Domain Differentially Respond to Load to Influence Drosophila Muscle Kinetics

2008-11-18

We measured the influence of alternative versions of the Drosophila melanogaster myosin heavy chain relay domain on muscle mechanical properties. We exchanged relay domain regions (encoded by alternative versions of exon 9) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin. Previously, we observed no effect of exchanging the EMB relay domain region into the flight muscle isoform (IFI-9b) on in vitro actin motility velocity or solution ATPase measurements compared to IFI. However, in indirect flight muscle fibers, IFI-9b exhibited decreased maximum power generation (P_max) and optimal frequency of power generation (f_max) to 70% and 83% of IFI fiber values. The decrease in muscle performance reduced the flight ability and wing-beat frequency of IFI-9b Drosophila compared to IFI Drosophila. Previously, we found that exchanging the flight muscle specific relay domain into the EMB isoform (EMB-9a) prevented actin movement in the in vitro motility assay compared to EMB, which does support actin movement. However, in indirect flight muscle fibers EMB-9a was a highly effective motor, increasing P_max and f_max 2.5-fold and 1.4-fold, respectively, compared to fibers expressing EMB. We propose that the oscillatory load EMB-9a experiences in the muscle fiber reduces a high activation energy barrier between two strongly bound states of the cross-bridge cycle, thereby promoting cross-bridge cycling. The IFI relay domain's enhanced sensitivity to load increases cross-bridge kinetics, whereas the EMB version is less load-sensitive.

[MUSCLE AND CONTRACTILITY] Structural Dynamics of the Actomyosin Complex Probed by a Bifunctional Spin Label that Cross-Links SH1 and SH2

Thompson, A. R., Naber, N., Wilson, C., Cooke, R., Thomas, D. D. — 2008-11-18

We have used a bifunctional spin label (BSL) to cross-link Cys⁷⁰⁷ (SH1) and Cys⁶⁹⁷ (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A·M·ADP·P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A·M·ADP. In contrast, helical order is observed for the strongly bound state A·M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A·M·ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A·M·ADP, slow rotational mobility comparable to that of A·M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.

[PROTEINS] Biophysical Study of Thermal Denaturation of Apo-Calmodulin: Dynamics of Native and Unfolded States

2008-11-18

Apo-calmodulin, a small, mainly , soluble protein is a calcium-dependent protein activator. This article presents a study of internal dynamics of native and thermal unfolded apo-calmodulin, using quasi-elastic neutron scattering. This technique can probe protein internal dynamics in the picosecond timescale and in the nanometer length-scale. It appears that a dynamical transition is associated with thermal denaturation of apo-calmodulin. This dynamical transition goes together with a decrease of the confinement of hydrogen atoms, a decrease of immobile protons proportion and an increase of dynamical heterogeneity. The comparison of native and unfolded states dynamics suggests that the dynamics of protein atoms is more influenced by their distance to the backbone than by their solvent exposure.

[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. — 2008-11-18

Atomistic molecular dynamics simulations are used to probe changes in the nature and subnanosecond dynamical behavior of solvation waters that accompany partial denaturation of the globular protein, human -lactalbumin. A simulated ensemble of subcompact conformers, similar to the molten globule state of human -lactalbumin, 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 compared to earlier experimental estimates in molten globule states, and is consistent with more recent descriptions based on nuclear magnetic relaxation dispersion studies.

[PROTEINS] An Altered Mode of Calcium Coordination in Methionine-Oxidized Calmodulin

Jones, E. M., Squier, T. C., Sacksteder, C. A. — 2008-11-18

Oxidation of methionine residues in calmodulin (CaM) lowers the affinity for calcium and results in an inability to activate target proteins fully. To evaluate the structural consequences of CaM oxidation, we used infrared difference spectroscopy to identify oxidation-dependent effects on protein conformation and calcium liganding. Oxidation-induced changes include an increase in hydration of -helices, as indicated in the downshift of the amide I' band of both apo-CaM 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 the model 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, suggesting that varying sensitivities of binding sites to oxidation may underlie the loss of CaM function upon oxidation.

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

McAllister, S. R., Floudas, C. A. — 2008-11-18

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.

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

Taniguchi, Y., Brockwell, D. J., Kawakami, M. — 2008-11-18

We 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 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, we observed a "hump-like" deviation due to the detachment of β-strand A from each I27 domain. The force at which the humps appear was ~130 pN and showed no temperature dependence, at least in the temperature range of 2°C–30°C. The hump structure was successfully analyzed 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 that the distance (x_u) 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 stabilized by topologically simple local hydrogen-bond network and that the temperature does not affect the detachment reaction thermodynamically and kinetically, whereas the interaction between the β-strands A' and G, which is a critical region for its mechanical stability, is strongly dependent on the temperature.

[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. — 2008-11-18

Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure resulted in a protein with fewer hemichrome impurities than was obtained by an 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 17–30 Å were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between ~30–140 K, and by spin-echo measurements of the enhancement of spin-spin relaxation rates at 10–30 K. Interspin distances were also calculated, using the molecular modeling program Insight II (Accelrys, San Diego, CA). For most 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 with spin-lattice relaxation enhancement, which permits measurements at longer distances.

[SUPRAMOLECULAR ASSEMBLIES] Nonexponential Kinetics of DNA Escape from {alpha}-Hemolysin Nanopores

Wiggin, M., Tropini, C., Tabard-Cossa, V., Jetha, N. N., Marziali, A. — 2008-11-18

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 nonexponential 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.

[SUPRAMOLECULAR ASSEMBLIES] Molecular Dynamics Simulation and Coarse-Grained Analysis of the Arp2/3 Complex

Pfaendtner, J., Voth, G. A. — 2008-11-18

A molecular dynamics investigation and coarse-grained analysis of inactivated actin-related protein (Arp) 2/3 complex is presented. It was found that the nucleotide binding site within Arp3 remained in a closed position with bound ATP or ADP, but opened when simulation with no nucleotide was performed. In contrast, simulation of the isolated Arp3 subunit with bound ATP, showed a fast opening of the nucleotide binding cleft. A homology model for the missing subdomains 1 and 2 of Arp2 was constructed, and it was also found that the Arp2 binding cleft remained closed with bound nucleotide. Within the nucleotide binding cleft a distinct opening and closing period of 10 ns was observed in many of the simulations of Arp2/3 as well as isolated Arp3. Substitution studies were employed, and several alanine substitutions were found to induce a partial opening of the ATP binding cleft in Arp3 and Arp2, whereas only a single substitution was found to induce opening of the ADP binding cleft. It was also found that the nucleotide type did not cause a substantial change on interfacial contacts between Arp3 and the ArpC2, ArpC3 and ArpC4 subunits. Nucleotide-free Arp3 had generally less stable contacts, but the overall contact architecture was constant. Finally, nucleotide-dependent coarse-grained models for Arp3 are developed that serve to further highlight the structural differences induced in Arp3 by nucleotide hydrolysis.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] A Method Improving the Accuracy of Fluorescence Recovery after Photobleaching Analysis

Jonsson, P., Jonsson, M. P., Tegenfeldt, J. O., Hook, F. — 2008-11-18

Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/noise ratio is low, when there are temporal fluctuations in the illumination, or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under nonideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within ~1%, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with <10% error.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Structural Refinement of Membrane Proteins by Restrained Molecular Dynamics and Solvent Accessibility Data

Sompornpisut, P., Roux, B., Perozo, E. — 2008-11-18

We present an approach for incorporating solvent accessibility data from electron paramagnetic resonance experiments in the structural refinement of membrane proteins through restrained molecular dynamics simulations. The restraints have been parameterized from oxygen (O₂) and nickel-ethylenediaminediacetic acid (NiEdda) collision frequencies, as indicators of lipid or aqueous exposed spin-label sites. These are enforced through interactions between a pseudoatom representation of the covalently attached Nitroxide spin-label and virtual "solvent" particles corresponding to O₂ and NiEdda in the surrounding environment. Interactions were computed using an empirical potential function, where the parameters have been optimized to account for the different accessibilities of the spin-label pseudoatoms to the surrounding environment. This approach, "pseudoatom-driven solvent accessibility refinement", was validated by refolding distorted conformations of the Streptomyces lividans potassium channel (KcsA), corresponding to a range of 2–30 Å root mean-square deviations away from the native structure. Molecular dynamics simulations based on up to 58 electron paramagnetic resonance restraints derived from spin-label mutants were able to converge toward the native structure within 1–3 Å root mean-square deviations with minimal computational cost. The use of energy-based ranking and structure similarity clustering as selection criteria helped in the convergence and identification of correctly folded structures from a large number of simulations. This approach can be applied to a variety of integral membrane protein systems, regardless of oligomeric state, and should be particularly useful in calculating conformational changes from a known reference crystal structure.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Macromolecular Crowding and Size Effects on Probe Microviscosity

Goins, A. B., Sanabria, H., Waxham, M. N. — 2008-11-18

Development of biologically relevant crowding solutions necessitates improved understanding of how the relative size and density of mobile obstacles affect probe diffusion. Both the crowding density and relative size of each co-solute in a mixture will contribute to the measured microviscosity as assessed by altered translational mobility. Using multiphoton fluorescent correlation spectroscopy, this study addresses how excluded volume of dextran polymers from 10 to 500 kDa affect microviscosity quantified by measurements of calmodulin labeled with green fluorescent protein as the diffusing probe. Autocorrelation functions were fit using both a multiple-component model with maximum entropy method (MEMFCS) and an anomalous model. Anomalous diffusion was not detected, but fits of the data with the multiple-component model revealed separable modes of diffusion. When the dominant mode of diffusion from the MEMFCS analysis was used, we observed that increased excluded volume slows probe mobility as a simple exponential with crowder concentration. This behavior can be modeled with a single parameter, β, which depends on the dextran size composition. Two additional modes of diffusion were observed using MEMFCS and were interpreted as unique microviscosities. The fast mode corresponded to unhindered free diffusion as in buffer, whereas the slower agreed well with the bulk viscosity. At 10% crowder concentration, one finds a microviscosity approximately three times that of water, which mimics that reported for intracellular viscosity.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Contact Guidance Mediated Three-Dimensional 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. — 2008-11-18

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 noninvading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/Rho kinase pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are prealigned, inhibiting neither Rho nor Rho kinase inhibits 3D migration, which supports our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Analysis of Molecular Concentration and Brightness from Fluorescence Fluctuation Data with an Electron Multiplied CCD Camera

Unruh, J. R., Gratton, E. — 2008-11-18

We demonstrate the calculation of particle brightness and concentration from fluorescence-fluctuation photon-counting statistics using an electron-multiplied charge-coupled device (EMCCD) camera. This technique provides a concentration-independent measure of particle brightness in dynamic systems. The high sensitivity and highly parallel detection of EMCCD cameras allow for imaging of dynamic particle brightness, providing the capability to follow aggregation reactions in real time. A critical factor of the EMCCD camera is the presence of nonlinearity at high intensities. These nonlinearities arise due to limited capacity of the CCD well and to the analog-to-digital converter maximum range. However, we show that the specific camera we used (with a 16-bit analog-to-digital converter) has sufficient dynamic range for most microscopy applications. In addition, we explore the importance of camera timing behavior as it is affected by the vertical frame transfer speed of the camera. Although the camera has microsecond exposure time for illumination of a few pixels, the exposure time increased to milliseconds for full-field illumination. Finally, we demonstrate the ability of the technique to follow concentration changes and measure single-molecule brightness in real time in living cells.

[SPECTROSCOPY, IMAGING, OTHER TECHNIQUES] Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy

2008-11-18

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.

[CELL BIOPHYSICS] Quantitative Measurement of cAMP Concentration Using an Exchange Protein Directly Activated by a cAMP-Based FRET-Sensor

Salonikidis, P. S., Zeug, A., Kobe, F., Ponimaskin, E., Richter, D. W. — 2008-11-18

Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a 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". 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 the 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 after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7).

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

Selhuber-Unkel, C., Lopez-Garcia, M., Kessler, H., Spatz, J. P. — 2008-11-18

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 arginine- glycine-aspartate 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 showed a reinforcement of adhesion if the distance between integrin molecules was <70 nm. This reinforcement had already occurred at cell-substrate contact times <5 min. We believe our results show 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 nanoscale.

[CELL BIOPHYSICS] Probing the Dynamic Organization of Transcription Compartments and Gene Loci within the Nucleus of Living Cells

Sinha, D. K., Banerjee, B., Maharana, S., Shivashankar, G. V. — 2008-11-18

The three-dimensional organization of nuclear compartments within living cells determines genome function and yet their underlying self-organizing principles are unclear. We visualize in real-time transcriptionally active compartments (TCs) by the transient enrichment of fluorescently-labeled uridine 5'-triphosphate molecules within living cells. These TCs partially colocalize with active RNA-Pol II in the cell nucleus. Fluorescence anisotropy maps of chromatin compaction evidences a more open chromatin structure at the TCs. Using live-cell timelapse imaging, heterogeneity in the dynamic behavior of TCs has been revealed which falls into three distinct classes: subdiffusive, super-diffusive, and normal diffusive behavior. In contrast, the mobility of a candidate gene locus, either in the repressed or activated state, undergoes a differential restricted motion that is coupled to TC movement. Further TC dynamics is directly affected by small molecule chromatin structure modulators and adenosine triphosphate depletion. This heterogeneous behavior in TC dynamics within living cells could provide an interesting paradigm to explore the spatiotemporal dimension to gene transcription control.

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

Zhang, Y., Sun, G., Lu, S., Li, N., Long, M. — 2008-11-18

Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although 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, how the low r_f (<10² pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap 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 the same r_f. The most probable force, f*, was enhanced with the spring constant when k < 47.0 x 10^–3 pN/nm, indicating that the bond dissociation at low r_f was spring constant dependent and that bond rupture force depended on both the loading rate and the mechanical compliance of force transducer. These results provide new insights into understanding the P-selectin glycoprotein ligand 1 bond dissociation at low r_f or k.

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

Stadler, A. M., Digel, I., Artmann, G. M., Embs, J. P., Zaccai, G., Buldt, G. — 2008-11-18

A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal 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 larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

[CELL BIOPHYSICS] Dysfunctional Connections Between the Nucleus and the Actin and Microtubule Networks in Laminopathic Models

2008-11-18

Laminopathies encompass a wide array of human diseases associated to scattered mutations along LMNA, a single gene encoding A-type lamins. How such genetic alterations translate to cellular defects and generate such diverse disease phenotypes remains enigmatic. Recent work has identified nuclear envelope proteins—emerin and the linker of the nucleoskeleton and cytoskeleton (LINC) complex—which connect the nuclear lamina to the cytoskeleton. Here we quantitatively examine the composition of the nuclear envelope, as well as the architecture and functions of the cytoskeleton in cells derived from two laminopathic mouse models, including Hutchinson-Gilford progeria syndrome (Lmna^L530P/L530P) and Emery-Dreifuss muscular dystrophy (Lmna^–/–). Cells derived from the overtly aphenotypical model of X-linked Emery-Dreifuss muscular dystrophy (Emd^–/y) were also included. We find that the centrosome is detached from the nucleus, preventing centrosome polarization in cells under flow—defects that are mediated by the loss of emerin from the nuclear envelope. Moreover, while basal actin and focal adhesion structure are mildly affected, RhoA activation, cell-substratum adhesion, and cytoplasmic elasticity are greatly lowered, exclusively in laminopathic models in which the LINC complex is disrupted. These results indicate a new function for emerin in cell polarization and suggest that laminopathies are not directly associated with cells' inability to polarize, but rather with cytoplasmic softening and weakened adhesion mediated by the disruption of the LINC complex across the nuclear envelope.

[CELL BIOPHYSICS] Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

Petrasek, Z., Hoege, C., Mashaghi, A., Ohrt, T., Hyman, A. A., Schwille, P. — 2008-11-18

The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

[RETRACTION] Retraction

2008-11-18