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Modeling the mechanisms of protofibril twisting: Molecular-dynamics simulations of simian viral peptide aggregates show that β sheets of 10 to 30 chains form left-handed helical ribbons with saddlelike curvature (see picture). These structures are highly dynamic, with oscillations around an average twist angle of 9-10°, and a pitch of 15-20 nm, depending on β-sheet length. The peptides studied are key to viral entry into host cells.

ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+) with each NBD indicates that the coordination of ATP and Mg(2+) differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations.

Sterols are the hallmarks of eukaryotic membranes where they are often found in specialized functional microdomains of the plasma membrane called lipid rafts. Despite some notable exceptions, prokaryotes lack sterols. However, growing evidence has suggested the existence of raft-like domains in the plasma membrane of bacteria. A structurally related family of triterpenoids found in some bacteria called hopanoids has long been assumed to be bacterial surrogates for sterols in membranes. Although the effect of sterols, in particular cholesterol, on lipid bilayers has been extensively characterized through experimental and simulation studies, those of hopanoids have hardly been investigated. In this study, molecular dynamics simulations are used to examine the effect of two hopanoids, diploptene (hop-22(29)-ene) and bacteriohopanetetrol ((32R,33S,34S)-bacteriohopane-32,33,34,35-tetrol), on a model bilayer. The results are compared with those obtained for cholesterol and a pure phosphatidylcholine bilayer. It is shown that diploptene and bacteriohopanetetrol behave very differently under the conditions simulated. Whereas bacteriohopanetetrol adopted a cholesterol-like upright orientation in the bilayer, diploptene partitioned between the two leaflets inside the bilayer. Analysis of various structural properties (area per lipid, electron density profile, tilt angle of the lipids, and conformation and order parameters of the phosphatidylcholine tails) in bacteriohopanetetrol- and cholesterol-containing bilayers indicates that the condensing and ordering effect of bacteriohopanetetrol is weaker than that of cholesterol. The simulations suggest that the chemical diversity of hopanoids may lead to a broader range of functional roles in bacterial membranes than sterols in eukaryotic membranes.

To test and validate the Automated force field Topology Builder and Repository (ATB; http://compbio.biosci.uq.edu.au/atb/ ) the hydration free enthalpies for a set of 214 drug-like molecules, including 47 molecules that form part of the SAMPL4 challenge have been estimated using thermodynamic integration and compared to experiment. The calculations were performed using a fully automated protocol that incorporated a dynamic analysis of the convergence and integration error in the selection of intermediate points. The system has been designed and implemented such that hydration free enthalpies can be obtained without manual intervention following the submission of a molecule to the ATB. The overall average unsigned error (AUE) using ATB 2.0 topologies for the complete set of 214 molecules was 6.7 kJ/mol and for molecules within the SAMPL4 7.5 kJ/mol. The root mean square error (RMSE) was 9.5 and 10.0 kJ/mol respectively. However, for molecules containing functional groups that form part of the main GROMOS force field the AUE was 3.4 kJ/mol and the RMSE was 4.0 kJ/mol. This suggests it will be possible to further refine the parameters provided by the ATB based on hydration free enthalpies.

This chapter discusses the study of the interaction of peptides and proteins at interfaces using molecular dynamics (MD) simulation techniques. First, the chapter discusses how computational methods, in particular MD simulation techniques, complement experimental methods. Then it focuses on three main areas of research: the interaction of peptides and proteins with (i) biological membranes and lipid bilayers, (ii) airâwater and oilâwater interfaces, and (iii) organic and inorganic sorbents.

We have investigated the feasibility of predicting free energy differences between a manifold of molecular states from a single simulation or ensemble representing one reference state. Two formulas that are based on the so-called λ- coupling parameter approach are analyzed and compared: (i) expansion of the free energy F(λ) into a Taylor series around a reference state (λ = 0), and (ii) the so-called free energy perturbation formula. The results obtained by these extrapolation methods are compared to exact (target) values calculated by thermodynamic integration for mutations in two molecular systems: a model dipolar diatomic molecule in water, and a series of para-substituted phenols in water. For moderate charge redistribution (≈0.5 e), both extrapolation methods reproduce the exact free energy differences. For free energy changes due to a change of atom type or size, the Taylor expansion method fails completely, while the perturbation formula yields moderately accurate predictions. Both extrapolation ...

Experimentally determined water/chloroform partition coefficients for three indole derivatives (3- methylindole, N-acetyltryptamine, and 3-(3'-indolyl)propionic acid N-methylamide), models of the amino acid tryptophan, are compared to free-energy differences calculated using molecular dynamics simulations. The effect of the choice of force field, the choice of pathway along which the free-energy change is calculated, the inclusion of free-energy contributions from constraints, and the treatment of long-range interactions on the agreement with the experimental data for this system are investigated with an eye to understanding the importance of the different aspects of the molecular model and computational procedure. It is demonstrated that, although the compounds are neutral and do not differ much in dipole moment or charge distribution, the incorporation of a reaction field to treat long-range electrostatic interactions is necessary to reproduce the experimentally observed trends. The implications of these findings for free-energy calculations in general and for the estimation of partition coefficients in particular are discussed.

Rapid non-empirical methods for estimating binding free energies are reviewed. A novel approach based on the application of the free energy perturbation formula to a biased ensemble is presented. Preliminary results demonstrating the applicability of this approach in protein systems are shown and the potential of this method in structure-based drug design is discussed.

The application of molecular dynamics computer simulation methods to study the dynamics of proteins is reviewed with an eye to its possibilities and limitations. Examples are given, mainly using nanosecond trajectories of the proteins bovine pancreatic trypsin inhibitor and lysozyme, of the different protein properties, of which the dynamics can be or cannot be sampled on a nanosecond time scale. It is concluded that the major asset of the simulation technique is that the different factors contributing to the dynamics of a particular process can be analyzed at atomic detail, as long as one has sampled the appropriate time scale.

A series of three molecular dynamics simulations at 300 K in explicit solvent environments of chloroform, methanol and water has been performed on the pulmonary surfactant lipoprotein, SP-C, comprising several consecutive valine residues in order to investigate the stability of the alpha-helical conformation. Two additional simulations were performed on truncated SP-C with a five-residue N-terminal deletion at 300 K and 500 K in water, the high temperature run in order to increase the rate of peptide denaturation. Indications of destabilization appear in chloroform during 1 ns while the SP-C alpha-helix is remarkably stable during 1 ns in methanol and water. In particular the polyvalyl part comprising residues Val15 to Val21 remains intact even at elevated temperature, and the valines do not disrupt the alpha-helical conformation. The valyl-rotamer sampling is partly restricted. Unfolding takes place successively along the primary sequence starting from the C-terminal end. Factors affecting polypeptide stability in molecular dynamics simulations are addressed. The intrinsic helix-forming tendency of valine residues and its dependence on the sequence context, and the role of the solvent environment in stabilizing or destabilizing an alpha-helical fold, are discussed.

Four methods are compared to drive the unfolding of a protein: (1) high tem- perature (T-run), (2) high pressure (P-run), (3) by imposing a gradual increase in the mean ra- dius of the protein using a penalty function added to the physical interaction function (F- run, radial force driven unfolding), and (4) by weak coupling of the difference between the temperature of the radially outward moving at- oms and the radially inward moving atoms to an external temperature bath (K-run, kinetic energy driven unfolding). The characteristic features of the four unfolding pathways are an- alyzed in order to detect distortions due to the size or the type of the applied perturbation, as well as the features that are common to all of them. Hen egg white lysozyme is used as a test system. The simulations are analyzed and com- pared to experimental data like 'H-NMR amide proton exchange-folding competition, heat ca- pacity, and compressibility measurements. 0 1995 Wiley-Liss, Inc.

Molecular dynamics simulations have been used to investigate the effect of DMSO on 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid bilayers. The concentration of DMSO was varied between 0 and 25.0 mol %. For both lipids, DMSO causes the membrane to expand in the plane of the membrane while thinning normal to that plane. Above a critical concentration, pores in the membrane form spontaneously, and if the concentration is increased further, then the bilayer structure is destroyed. Even at concentrations below those required to induce pores, DMSO readily diffuses across the bilayers. The free-energy profile associated with the diffusion of a DMSO molecules across the membrane has been calculated. The simulations suggest that the DOPC bilayer is more resistant to the deleterious effects of DMSO, both increasing the stability of the membranes and decreasing the rate at which DMSO diffuses across the membrane. In this way, the work highlights the importance of investigating the lipid composition of cell membranes when characterizing the effects of cryosolvents.

Biofilm formation, in which bacteria are embedded within an extracellular matrix, is the default form of microbial life in most natural and engineered habitats. In this work, atomistic molecular dynamics simulations were employed to examine the self-assembly of the polysaccharide Granulan to provide insight into the molecular interactions that lead to biofilm formation. Granulan is a major gel forming matrix component of granular microbial biofilms found in used-water treatment systems. Molecular dynamics simulations showed that Granulan forms an antiparallel double helix stabilized by complementary hydrogen bonds between the β-glucosamine of one strand and the N-acetyl-β-galactosamine-2-acetoamido-2-deoxy-α-galactopyranuronic pair of the other in both the presence and absence of Ca(2+). It is shown that Ca(2+) binds primarily to the carboxyl group of the terminal hexuronic acid of the sugar branch and that interactions between branches mediated by Ca(2+) suggest a possible mechanism for strengthening gels by facilitating interhelical bridging.

By considering all possible mutations among four para-substituted phenols, p-chlorophenol, p-methylphenol, p-cyanophenol, and p-methoxyphenol, which bind as inclusion compounds in alpha-cyclodextrin, the convergence properties of thermodynamic integration free energy calculations using slow growth as compared to numerical quadrature are investigated and interpreted in terms of structural and dynamical properties of the molecular system. It is shown that a systematic increase in the calculated hysteresis can be expected with increasing simulation time in slow-growth calculations if the system is perturbed faster than the rate at which the various states that make up the equilibrium ensemble are sampled. Using numerical quadrature the effects of nonequilibrium can be largely separated from the effects of insufficient sampling. It is shown, however, that the apparent degree of convergence when using numerical quadrature does not necessarily reflect the accuracy of the calculation. The utility of formulating closed cycles in both the bound and unbound states as a means of determining the minimum error in a given calculation is demonstrated. The effects of the choice of pathway and of the choice of integration scheme on convergence within closed cycles are also discussed. Finally, the quality of the force field used and the relative importance of the force field as opposed to sampling considerations are assessed by comparing the estimated free energy differences to experimental data. It is shown that a meaningful appraisal of a specific force field cannot be made independent of sampling considerations. A modification to the GROMOS force field that improved the agreement between the calculated and experimental free energies for the mutation of p-chlorophenol to p-methylphenol is also proposed.

Recently, a number of methods have been proposed that are designed to extract contributions to the change in free energy associated with a given perturbation or mutation of a protein originating from specific residue-residue or atom-atom interactions, both based on theoretical calculations and on experimental data. We caution here that detailed analysis based on these methods is unreliable. It is demonstrated, both from first principles using statistical mechanics and by way of example, that in a general case a meaningful decomposition of the free energy in terms of specific residue-residue or atom-atom interactions is not possible.

A simple, general and numerically stable approach for avoiding the singularities which generally occur when atoms or interaction sites are created or annihilated in free energy calculations based on computer simulations is presented. The origin of such singularities and numerical instabilities occurring in Monte Carlo or molecular dynamics simulations is discussed, as is the limited accuracy of the techniques currently used to avoid such difficulties.

The efficiency of using a variant of Hamiltonian replica-exchange molecular dynamics (Chaperone H-replica-exchange molecular dynamics [CH-REMD]) for the refinement of protein structural models generated de novo is investigated. In CH-REMD, the interaction between the protein and its environment, specifically, the electrostatic interaction between the protein and the solvating water, is varied leading to cycles of partial unfolding and refolding mimicking some aspects of folding chaperones. In 10 of the 15 cases examined, the CH-REMD approach sampled structures in which the root-mean-square deviation (RMSD) of secondary structure elements (SSE-RMSD) with respect to the experimental structure was more than 1.0 Å lower than the initial de novo model. In 14 of the 15 cases, the improvement was more than 0.5 Å. The ability of three different statistical potentials to identify near-native conformations was also examined. Little correlation between the SSE-RMSD of the sampled structures with respect to the experimental structure and any of the scoring functions tested was found. The most effective scoring function tested was the DFIRE potential. Using the DFIRE potential, the SSE-RMSD of the best scoring structures was on average 0.3 Å lower than the initial model. Overall the work demonstrates that targeted enhanced-sampling techniques such as CH-REMD can lead to the systematic refinement of protein structural models generated de novo but that improved potentials for the identification of near-native structures are still needed.

The relative binding free energy between two ligands to a specific protein can be obtained using various computational methods. The more accurate and also computationally more demanding techniques are the so-called free energy methods which use conformational sampling from molecular dynamics or Monte Carlo simulations to generate thermodynamic averages. Two such widely applied methods are the thermodynamic integration (TI) and the recently introduced enveloping distribution sampling (EDS) methods. In both cases relative binding free energies are obtained through the alchemical perturbations of one ligand into another in water and inside the binding pocket of the protein. TI requires many separate simulations and the specification of a pathway along which the system is perturbed from one ligand to another. Using the EDS approach, only a single automatically derived reference state enveloping both end states needs to be sampled. In addition, the choice of an optimal pathway in TI calculations is not trivial and a poor choice may lead to poor convergence along the pathway. Given this, EDS is expected to be a valuable and computationally efficient alternative to TI. In this study, the performances of these two methods are compared using the binding of ten tetrahydroisoquinoline derivatives to phenylethanolamine N-transferase as an example. The ligands involve a diverse set of functional groups leading to a wide range of free energy differences. In addition, two different schemes to determine automatically the EDS reference state parameters and two different topology approaches are compared.

European Biophysics Journal, 40 (7), ISSN:0175-7571, ISSN:1432-1017

The partition coefficient of cyclosporin A (CsA) was measured in octanol/water and heptane/water by centrifugal partition chromatography. By comparison with results from model compounds, it was deduced that the hydrogen-bonding capacity of CsA changed dramatically from an apolar solvent (where it is internally H-bonded) to polar solvents (where it exposes its H-bonding groups to the solvent). Molecular dynamics simulations in water and CCl4 support the suggestion that CsA undergoes a solvent-dependent conformational changes and that the interconversion process is slow on the molecular dynamics time scale.

The static and frequency dependent dielectric properties of trypsin inhibitor and hen egg white lysozyme have been calculated from the fluctuations in the total dipole moment of the two proteins. Total dipole moment fluctuations were obtained from long molecular dynamics simulations of both proteins in an explicit solvent environment. Despite differences in the total charge, volume, shape and secondary structure composition of the two proteins, consistent values for the dielectric constant were obtained. The static dielectric constant of trypsin inhibitor was calculated to be 36, compared with a value of 30 for hen egg white lysozyme. Convergence in the calculations required 1 ns of simulation. The calculated frequency dependent dielectric constant was also consistent with known experimental dielectric dispersion curves for proteins in aqueous solution. The implications for free energy calculations involving significant charge redistribution are also discussed.

Poly(ethylene glycol) (PEG) is used as an inert spacer in a wide range of biotechnological applications such as to display peptides and proteins on surfaces for diagnostic purposes. In such applications it is critical that the peptide is accessible to solvent and that the PEG does not affect the conformational properties of the peptide to which it is attached. Using molecular dynamics (MD) simulation techniques, we have investigated the influence of a commonly used PEG spacer on the conformation properties of a series of five peptides with differing physical-chemical properties (YGSLPQ, VFVVFV, GSGGSG, EEGEEG, and KKGKKG). The conformational properties of the peptides were compared (a) free in solution, (b) attached to a PEG-11 spacer in solution, and (c) constrained to a two-dimensional lattice via a (PEG-11)(3) spacer, mimicking a peptide displayed on a surface as used in microarray techniques. The simulations suggest that the PEG spacer has little effect on the conformational properties of small neutral peptides but has a significant effect on the conformational properties of small highly charged peptides. When constrained to a two-dimensional surface at peptide densities similar to those used experimentally, it was found that the peptides, in particular the polar and nonpolar peptides, aggregated strongly. The peptides also partitioned into the PEG layer. Potentially, this means that at high packing densities only a small fraction of the peptide attached to the surface would in fact be accessible to a potential interaction partner.

Despite its central role in structure based drug design the determination of the binding mode (position, orientation and conformation in addition to protonation and tautomeric states) of small heteromolecular ligands in protein:ligand complexes based on medium resolution X-ray diffraction data is highly challenging. In this perspective we demonstrate how a combination of molecular dynamics simulations and free energy (FE) calculations can be used to correct and identify thermodynamically stable binding modes of ligands in X-ray crystal complexes. The consequences of inappropriate ligand structure, force field and the absence of electrostatics during X-ray refinement are highlighted. The implications of such uncertainties and errors for the validation of virtual screening and fragment-based drug design based on high throughput X-ray crystallography are discussed with possible solutions and guidelines.

The continuous advance of experimental techniques is steadily increasing our knowledge of biochemical systems and processes. A detailed picture of many biomolecular processes is emerging due to the possibility of measuring atomic properties of biological macromolecules, such as proteins. X-ray diffraction has provided a three-dimensional picture of biomolecular assemblies of ever increasing size, ranging from small proteins to a complete macromolecular reaction centre. Other experimental techniques, like nuclear magnetic resonance (NMR) and other spectroscopic methods, yield less complete information at the atomic level. However, the development of multi-dimensional NMR has made it possible to determine the spatial structure of small proteins, albeit at low resolution. The advantage of spectroscopic measuring techniques over X-ray diffraction methods is that the former can be used to obtain information on the dynamics of specific atoms or groups of atoms in the biomolecule, while the latter only yield an indication of the mobility of atoms, not of the time scale of their motion. The other major step forward is the advent of the possibility to change the amino acid composition of a protein at will. Instead of studying the proteins with which nature has provided us, we may now make specific mutations and study their effect on protein properties.

Resorcinolic lipids, or resorcinols, are commonly found in plant membranes. They consist of a substituted benzene ring forming the hydrophilic lipid head, attached to an alkyl chain forming the hydrophobic tail. Experimental results show alternative effects of resorcinols on lipid membranes. Depending on whether they are added to lipid solutions before or after the formation of the liposomes, they either stabilize or destabilize these liposomes. Here we use atomistic molecular dynamics simulations to elucidate the molecular nature of this dual effect. Systems composed of either one of three resorcinol homologs, differing in the alkyl tail length, interacting with dimyristoylphosphatidylcholine lipid bilayers were studied. It is shown that resorcinols preincorporated into bilayers induce order within the lipid acyl chains, decrease the hydration of the lipid headgroups, and make the bilayers less permeable to water. In contrast, simulations in which the resorcinols are incorporated from the aqueous solution into a preformed phospholipid bilayer induce local disruption, leading to either transient pore formation or even complete rupture of the membrane. In line with the experimental data, our simulations thus demonstrate that resorcinols can either disturb or stabilize the membrane structure, and offer a detailed view of the underlying molecular mechanism.

A combination of MD simulations and free energy calculations have been used to propose a new model for the binding of amino acids to threonyl-tRNA-synthetase which not only yields a stable binding mode for l-Ser but also can explain the mechanism by which the editing domains of aminoacyl-tRNA-synthetases are enantiomeric selective preferentially binding d-amino acids.

The purple membrane (PM) is a specialized membrane patch found in halophilic archaea, containing the photoreceptor bacteriorhodopsin (bR). It is long known that calcium ions bind to the PM, but their position and role remain elusive to date. Molecular dynamics simulations in conjunction with a highly detailed model of the PM have been used to investigate the stability of calcium ions placed at three proposed cation binding sites within bR, one near the Schiff base, one in the region of the proton release group, and one near Glu9. The simulations suggest that, of the sites investigated, the binding of calcium ions was most likely at the proton release group. Binding in the region of the Schiff base, while possible, was associated with significant changes in local geometry. Calcium ions placed near Glu9 in the interior of bR (simultaneously to a Ca(2+) near the Schiff base and another one near the Glu194-Glu204 site) were not stable. The results obtained are discussed in relation to recent experimental observations and theoretical considerations.

Methods to compute free energy differences between different states of a molecular system are reviewed with the aim of identifying their basic ingredients and their utility when applied in practice to biomolecular systems. A free energy calculation is comprised of three basic components: (i) a suitable model or Hamiltonian, (ii) a sampling protocol with which one can generate a representative ensemble of molecular configurations, and (iii) an estimator of the free energy difference itself. Alternative sampling protocols can be distinguished according to whether one or more states are to be sampled. In cases where only a single state is considered, six alternative techniques could be distinguished: (i) changing the dynamics, (ii) deforming the energy surface, (iii) extending the dimensionality, (iv) perturbing the forces, (v) reducing the number of degrees of freedom, and (vi) multi-copy approaches. In cases where multiple states are to be sampled, the three primary techniques are staging, importance sampling, and adiabatic decoupling. Estimators of the free energy can be classified as global methods that either count the number of times a given state is sampled or use energy differences. Or, they can be classified as local methods that either make use of the force or are based on transition probabilities. Finally, this overview of the available techniques and how they can be best used in a practical context is aimed at helping the reader choose the most appropriate combination of approaches for the biomolecular system, Hamiltonian and free energy difference of interest. © 2009 Wiley Periodicals, Inc.

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.

A general method is presented to reduce the simulation time required to compute the relative free energy between two states X and Y of a molecular system by computer simulation. Although the free energy difference DELTA-A(x-->y) is, in principle, independent of the pathway chosen to change X into Y, in practice its choice strongly affects the accuracy of the obtained DELTA-A value. The optimum path is the one for which the relaxation time of the system tau-system attains a minimum, allowing the system to remain as close as possible near equilibrium during a simulation. Downscaling the relevant parts of the potential energy function before the change from X to Y is made, and upscaling afterwards is a rather general way to shorten tau-system and thus save computing time. For a model system of butane like molecules the proposed procedure is more than 1 order of magnitude more efficient than the conventional technique of direct interconversion from state X to state Y.

Penrose and Hameroff have argued that the conventional models of a brain function based on neural networks alone cannot account for human consciousness, claiming that quantum-computation elements are also required. Specifically, in their Orchestrated Objective Reduction (Orch OR) model [R. Penrose and S. R. Hameroff, J. Conscious. Stud. 2, 99 (1995)], it is postulated that microtubules act as quantum processing units, with individual tubulin dimers forming the computational elements. This model requires that the tubulin is able to switch between alternative conformational states in a coherent manner, and that this process be rapid on the physiological time scale. Here, the biological feasibility of the Orch OR proposal is examined in light of recent experimental studies on microtubule assembly and dynamics. It is shown that the tubulins do not possess essential properties required for the Orch OR proposal, as originally proposed, to hold. Further, we consider also recent progress in the understanding of the long-lived coherent motions in biological systems, a feature critical to Orch OR, and show that no reformation of the proposal based on known physical paradigms could lead to quantum computing within microtubules. Hence, the Orch OR model is not a feasible explanation of the origin of consciousness.

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.

Aufbauend auf bekannten physikochemischen Eigenschaften des Insulins in Losung sowie auf bereits veroffentlichten Modellen wurden vier Selbstassoziationsmodelle ausgewahlt. uber einen weiten Bereich experimenteller Bedingungen (pH 2,7 und 10, Ionenstarke 0.05 und 0.1, t = 25 und 37 °C) wurden die Konzentrationsverteilungen von zinkfreiem Insulin gemessen und mit den Modellen mit und ohne Korrektur fur nicht-ideales Verhalten verglichen. Ein Vergleich der Kurven zeigt, das drei der Modelle als zufriedenstellende Beschreibungen der Gleichgewichte des Insulinsystems in wasrigen Losungen angesehen werden mussen. Signifikante Unterschiede, wie auch Gemeinsamkeiten dieser Modelle werden durch einen Vergleich der Verteilung monomerer und polymerer Formen bei drei verschiedenen Insulinkonzentrationen abgeschatzt. Die Konzentrationen wurden nach praktischer Nutzlichkeit gewahlt: Serumkonzentrationen, pharmakologische und physikochemische Konzentrationen. Der Wert der Protein-Selbstassoziationsanalyse wird mit Hilfe von Gleichungen diskutiert, die fur jeweils bestimmte Modelle erstellt werden, wobei besonders auf die Messungen des osmotischen Drucks Bezug genommen wird, die von anderen an Insulin-Varianten durchgefuhrt wurden, die gentechnisch zu monomeren Formen modifiziert wurden.

Computer-based molecular simulation techniques are increasingly used to interpret experimental data on biomolecular systems at an atomic level. Direct comparison between experiment and simulation is, however, seldom straightforward. The available experimental data are limited in scope and generally correspond to averages over both time and space. A critical analysis of the various factors that may influence the apparent degree of agreement between the results of simulations and experimentally measured quantities is presented and illustrated using examples from recent literature.

The sensitivity of molecular dynamics simulations to variations in the force field has been examined in relation to a set of 36 structures corresponding to 31 proteins simulated by using different versions of the GROMOS force field. The three parameter sets used (43a1, 53a5, and 53a6) differ significantly in regard to the nonbonded parameters for polar functional groups and their ability to reproduce the correct solvation and partitioning behavior of small molecular analogues of the amino acid side chains. Despite the differences in the force field parameters no major differences could be detected in a wide range of structural properties such as the root-mean-square deviation from the experimental structure, radii of gyration, solvent accessible surface, secondary structure, or hydrogen bond propensities on a 5 to 10 ns time scale. The small differences that were observed correlated primarily with the presence of charged residues as opposed to residues that differed most between the parameter sets. The work highlights the variation that can be observed in nanosecond simulations of protein systems and implications of this for force field validation, as well as for the analysis of protein simulations in general.

Three host-guest systems have been characterized using surface tension (sigma), calorimetry, and molecular dynamics simulations (MD). The hosts were three native cyclodextrins (CD) and the guest the non-ionic carbohydrate surfactant octyl-beta-d-glucopyranoside. It is shown that, for any host-guest system, a rough screening of the most probable complex stoichiometries can be obtained in a model free form, using only calorimetric data. The sigma data were analyzed using a model that includes a newly proposed adsorption isotherm. The equilibrium constants for several stoichiometries were simultaneously obtained through fitting the sigma data. For alpha- and beta-CD, the predominant species is 1:1 and to a lesser extent 2:1, disregarding the existence of the 1:2. For gamma-CD, the 1:2 species dominates, the other two being also present. In an attempt to confirm these results, 10 ns MD simulations for each CD were performed using seven different starting conformations. The MD stable conformations agree with the results found from the experimental data. In one case, the spontaneous dissociation-formation of a complex was observed. Analysis of the trajectories indicates that hydrophobic interactions are primarily responsible for the formation and stability of the inclusion complexes. For the 2:1 species, intermolecular H-bonds between CD molecules result in a tight packed structure where their original truncated cone shape is lost in favor of a cylindrical geometry. Together, the results clearly demonstrate that the often used assumption of considering only a 1:1 species is inappropriate.

Replica exchange methods REMs are increasingly used to improve sampling in molecular dynamics MD simulations of biomolecular systems. However, despite having been shown to be very effective on model systems, the application of REM in complex systems such as for the simulation of protein and peptide folding in explicit solvent has not been objectively tested in detail. Here we present a comparison of conventional MD and temperature replica exchange MD T-REMD simulations of a -heptapeptide in explicit solvent. This system has previously been shown to undergo reversible folding on the time scales accessible to MD simulation and thus allows a direct one-to-one comparison of efficiency. The primary properties compared are the free energy of folding and the relative populations of different conformers as a function of temperature. It is found that to achieve a similar degree of precision T-REMD simulations starting from a random set of initial configurations were approximately an order of magnitude more computationally efficient than a single 800 ns conventional MD simulation for this system at the lowest temperature investigated 275 K. However, whereas it was found that T-REMD simulations are more than four times more efficient than multiple independent MD simulations at one temperature 300 K the actual increase in conformation sampling was only twofold. The overall gain in efficiency using REMD resulted primarily from the ordering of different conformational states over temperature, as opposed to a large increase of conformational sampling. It is also shown that in this system exchanges are accepted primarily based on random fluctuations within the solvent and are not strongly correlated with the instantaneous peptide conformation raising questions in regard to the efficiency of T-REMD in larger systems. © 2007 American Institute of Physics. DOI: 10.1063/1.2404954

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Macromolecular Models: From Theories to Effective Algorithms.- Membrane Protein Simulations: Modelling a Complex Environment.- Modeling and Simulation Based Approaches for Investigating Allosteric Regulation in Enzymes.- Exploring the Connection Between Synthetic and Natural RNAs in Genomes: A Novel Computational Approach.- Learning to Align Sequences: A Maximum-Margin Approach.- Minimization of Complex Molecular Landscapes.- Overcoming Energetic and Time Scale Barriers Using the Potential Energy Surface.- The Protein Folding Problem.- Dynamical and Stochastic-Dynamical Foundations for Macromolecular Modelling.- Biomolecular Sampling: Algorithms, Test Molecules, and Metrics.- Approach to Thermal Equilibrium in Biomolecular Simulation.- The Targeted Shadowing Hybrid Monte Carlo (TSHMC) Method.- The Langevin Equation for Generalized Coordinates.- Metastability and Dominant Eigenvalues of Transfer Operators.- Computation of the Free Energy.- Free Energy Calculations in Biological Systems. How Useful Are They in Practice?.- Numerical Methods for Calculating the Potential of Mean Force.- Replica-Exchange-Based Free-Energy Methods.- Fast Electrostatics and Enhanced Solvation Models.- Implicit Solvent Electrostatics in Biomolecular Simulation.- New Distributed Multipole Metdhods for Accurate Electrostatics in Large-Scale Biomolecular Simulations.- Quantum-Chemical Models for Macromolecular Simulation.- Towards Fast and Reliable Quantum Chemical Modelling of Macromolecules.- Quantum Chemistry Simulations of Glycopeptide Antibiotics.- Panel Discussion.

We describe molecular dynamics simulations elucidating the molecular details of the process of fusion for small lipid vesicles. The simulations are based on a coarse grained (CG) lipid model that accurately represents the lamellar state of a variety of phospholipids and enables us to observe intermediate stages during fusion at near atomic detail. Simulations were conducted on a variety of systems containing common phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysoPC, and mixtures of the above. The fusion intermediates found are in general agreement with the stalk-pore mechanism. Transient pores sometimes form adjacent to the stalk, however, resulting in the mixing of lipids from the outer and inner monolayers. The speed of stalk formation and the opening of the fusion pore can be modulated by altering the lipid composition in qualitative agreement with experimental observations.

We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modi. cation of two hydrophobic core positions by Phe/[N-15,C-13]Leu enable the study of the folding characteristics and of distinct amino acid side chain interactions by fluorescence resonance energy transfer (FRET) and NMR spectroscopy. Results of both experiments reveal the antiparallel alignment of the helices and thus prove the design concept. This finding is also supported by molecular dynamics simulations. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in combination with NMR experiments was used for veri. cation of the oligomerization equilibria of the coiled coil peptide.

Molecular dynamics simulations have been used to study the spontaneous aggregation of a concentrated solution of dipalmitoylphosphatidylcholine (DPPC) molecules in water into a small vesicle. The molecules were represented in atomistic detail. Starting from a DPPC solution in water, an oblong vesicle with a long axis of 15 nm and short axes of 10 nm was formed spontaneously. After 90 ns of simulation, the vesicle contained a number of water pores. Water pores were shown to facilitate exchange of lipids between inner and outer leaflets. Lipid tails were shown to be less ordered in the inner leaflet of the vesicle, as compared to those in the outer leaflet of the vesicle and an equilibrated lamellar bilayer.

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.

The use of classical molecular dynamics simulations, performed in explicit water, for the refinement of structural models of proteins generated ab initio or based on homology has been investigated. The study involved a test set of 15 proteins that were previously used by Baker and coworkers to assess the efficiency of the ROSETTA method for ab initio protein structure prediction. For each protein, four models generated using the ROSETTA procedure were simulated for periods of between 5 and 400 nsec in explicit solvent, under identical conditions. In addition, the experimentally determined structure and the experimentally derived structure in which the side chains of all residues had been deleted and then regenerated using the WHATIF program were simulated and used as controls. A significant improvement in the deviation of the model structures from the experimentally determined structures was observed in several cases. In addition, it was found that in certain cases in which the experimental structure deviated rapidly from the initial structure in the simulations, indicating internal strain, the structures were more stable after regenerating the side-chain positions. Overall, the results indicate that molecular dynamics simulations on a tens to hundreds of nanoseconds time scale are useful for the refinement of homology or ab initio models of small to medium-size proteins.

The relative stability of protein structures determined by either X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy has been investigated by using molecular dynamics simulation techniques. Published structures of 34 proteins containing between 50 and 100 residues have been evaluated. The proteins selected represent a mixture of secondary structure types including all alpha, all beta, and alpha/beta. The proteins selected do not contain cysteine-eysteine bridges. In addition, any crystallographic waters, metal ions, cofactors, or bound ligands were removed before the systems were simulated. The stability of the structures was evaluated by simulating, under identical conditions, each of the proteins for at least 5 ns in explicit solvent. It is found that not only do NMR-derived structures have, on average, higher internal strain than structures determined by X-ray crystallography but that a significant proportion of the structures are unstable and rapidly diverge in simulations. Proteins 2003;52:111-120. (C) 2003 Wiley-Liss, Inc.

We report results from molecular dynamics simulations of the freezing and melting, at ambient temperature (T=300 K), of a bilayer of liquid water induced by either changing the distance between two confining parallel walls at constant lateral pressure or by lateral compression at constant plate separation. Both transitions are found to be first order. The system studied consisted of 1200 water molecules that were described by the TIP5P model. The in-plane symmetry of the oxygen atoms in the ice bilayer was found to be rhombic with a distorted in-registry arrangement. Above and below the stability region of the ice bilayer we observed two bilayer phases of liquid water that differ in the local ordering at the level of the second shell of nearest neighbors and in the density profile normal to the plane, yielding two liquid phases with different densities. These results suggest the intriguing possibility of a liquid-liquid transition of water, confined to a bilayer, at regions where the ice bilayer is unstable with respect to either of the liquid phases. In addition, we find that under the same conditions, water confined to 3-8 layers remains in the liquid phase (albeit stratification of the transverse density profile) with values of the lateral diffusion coefficient comparable to that of the bulk. (C) 2003 American Institute of Physics.

We report results from molecular dynamics simulations of water under confinement and at ambient conditions that predict a first-order freezing transition from a monolayer of liquid water to a monolayer of ice induced by increasing the distance between the confining parallel plates. Since a slab geometry is incompatible with a tetrahedral arrangement of the sp(3) hybridized oxygen of water, the freezing is coupled to a linear buckling transition. By exploiting the ordered out-of-plane displacement of the molecules in the buckled phase the distortion of the hydrogen bonds is minimized.

Molecular dynamics simulations of pore formation and mem-brane rupture in phospholipid bilayers under mechanical andelectrical stress at an atomic level are presented. Pore formationcan be induced on a nanosecond time scale in simulations wherethe lateral pressure exceeds -200 bar or where an electric field of0.5 V/nm is applied across the membrane.Lipid bilayer membranes are remarkable structures consistingof two leaflets of phospholipids. Their mechanical properties arecentral to understanding the behavior of cell membranes. Forexample, the formation of transient water pores is believed tounderlie the passive transport of protons and hydrophilic compoundsthrough the bilayer. Pore formation is also relevant during processessuch as cell fusion and is important for drug release from liposomes.Experimentally, pores can be induced in membranes by applyingmechanical stress (i.e., pipet aspiration experiments) or an electricfield (electroporation). The precise mechanism by which pores formand their size, structure, and stability are, however, poorlyunderstood.