Structure of the lipodepsipeptide syringomycin E in phospholipids and sodium dodecylsulphate micelle studied by circular dichroism, NMR spectroscopy and molecular dynamics.

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.

Molecular origin of Gerstmann-Sträussler-Scheinker syndrome: insight from computer simulation of an amyloidogenic prion peptide.

Prion proteins become pathogenic through misfolding. Here, we characterize the folding of a peptide consisting of residues 109-122 of the Syrian hamster prion protein (the H1 peptide) and of a more amyloidogenic A117V point mutant that leads in humans to an inheritable form of the Gerstmann-Sträussler-Scheinker syndrome. Atomistic molecular dynamics simulations are performed for 2.5 µs. Both peptides lose their α-helical starting conformations and assume a ß-hairpin that is structurally similar in both systems. In each simulation several unfolding/refolding events occur, leading to convergence of the thermodynamics of the conformational states to within 1 kJ/mol. The similar stability of the ß-hairpin relative to the unfolded state is observed in the two peptides. However, substantial differences are found between the two unfolded states. A local minimum is found within the free energy unfolded basin of the A117V mutant populated by misfolded collapsed conformations of comparable stability to the ß-hairpin state, consistent with increased amyloidogenicity. This population, in which V117 stabilizes a hydrophobic core, is absent in the wild-type peptide. These results are supported by simulations of oligomers showing a slightly higher stability of the associated structures and a lower barrier to association for the mutated peptide. Hence, a single point mutation carrying only two additional methyl groups is here shown to be responsible for rather dramatic differences of structuring within the unfolded (misfolded) state.

The effects of the L29F mutation on the ligand migration kinetics in crystallized myoglobin as revealed by molecular dynamics simulations.

By using multiple molecular dynamics (MD) trajectories, a quantitative description of carbon monoxide (CO) migration within crystal of L29F myoglobin mutant (L29F-Mb) was obtained. The aim was to provide a detailed model for ligand diffusion in the protein to be compared to the available L29F-Mb experimental-computational data and to the corresponding model kinetics we previously obtained for photolyzed CO within crystallized wild-type myoglobin (wt-Mb). Results suggest a clear migration pathway from distal pocket to the proximal site, similar to the one observed in wt-Mb, with a relaxation kinetics differing from the wt-Mb one essentially for the escape rate which is much higher in the mutant. Moreover MD data indicated a clear correlation between CO location within the protein and the conformation adopted by Phe29, well matching the available experimental data as obtained by time-resolved X-ray density maps. Such data, further validating the model used in the simulations, point out the subtle mutual effect between ligand diffusion and protein functional motions possibly explaining the observed dramatic variation of CO exit rate in L29F-Mb.

Conformational study of bovine lactoferricin in membrane-micking conditions by molecular dynamics simulation and circular dichroism.

Lactoferricins are potent antimicrobial peptides released by pepsin cleavage of Lactoferrins. Bovine Lactoferricin (LfcinB) has higher activity than the intact bovine Lactoferrin, and is the most active among the other Lactoferricins of human, murine and caprine origin. In the intact protein the fragment corresponding to LfcinB is in an helical conformation, while in water LfcinB adopts an amphipathic ß-hairpin structure. However, whether any of these structural motifs is the antibacterial active conformation, i.e., the one interacting with bacterial membrane components, remains to be seen. Here we present Circular Dichroism (CD) spectra and Molecular Dynamics (MD) simulations indicating that in membrane-mimicking solvents the LfcinB adopts an amphipathic ß-hairpin structure similar to that observed in water, but differing in the dynamic behavior of the side-chains of the two tryptophan residues. In the membrane-mimicking solvent these side-chains show a high propensity to point towards the hydrophobic environment, rather than being in the hydrophobic core as seen in water, while the backbone preserves the hairpin conformation as found in water. These results suggest that the tryptophans might act as anchors pulling the stable, solvent-invariant hairpin structure into the membrane.

Dynamic investigation of protein metal active sites: interplay of XANES and molecular dynamics simulations.

The effect of structural disorder on the X-ray absorption near-edge structure (XANES) spectrum of a heme protein has been investigated using the dynamical description of the system derived from molecular dynamics (MD) simulations. The XANES spectra of neuroglobin (Ngb) and carbonmonoxy-neuroglobin (NgbCO) have been quantitatively reproduced, starting from the MD geometrical configurations, without carrying out any optimization in the structural parameter space. These results provide an important experimental validation of the reliability of the potentials used in the MD simulations and accordingly corroborate the consistency of the structural dynamic information on the metal center, related to its biological function. This analysis allowed us to demonstrate that the configurational disorder associated with the distortion of the heme plane and with the different orientations of the axial ligands can affect the XANES features at very low energy. Neglecting configurational disorder in the XANES quantitative analysis of heme proteins is a source of systematic errors in the determination of Fe coordination geometry. The combined use of XANES and MD is a novel strategy to enhance the resolution and reliability of the structural information obtained on metalloproteins, making the combination of these techniques powerful for metalloprotein investigations.

The kinetics of ligand migration in crystallized myoglobin as revealed by molecular dynamics simulations.

By using multiple molecular dynamics trajectories of photolyzed carbon monoxide (CO) within crystallized myoglobin, a quantitative description of CO diffusion and corresponding kinetics was obtained. Molecular dynamics results allowed us to construct a detailed kinetic model of the migration process, shedding light on the kinetic mechanism and relevant steps of CO migration and remarkably-well reproducing the available experimental data as provided by time-resolved Laue x-ray diffraction.

Molecular dynamics simulation of the neuroglobin crystal: comparison with the simulation in solution.

Neuroglobin (Ngb) is a monomeric protein that, despite the small sequence similarity with other globins, displays the typical globin fold. In the absence of exogenous ligands, the ferric and the ferrous forms of Ngb are both hexacoordinated to the distal and proximal histidines. In the ferrous form, oxygen, nitric oxide or carbon monoxide can displace the distal histidine, yielding a reversible adduct. Crystallographic data show that the binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities. Molecular dynamics (MD) simulations in solution show that the heme oscillates between two positions, much as the ones observed in the crystal structure, although the occupancy is different. The simulations also suggest that ligand binding in solution can affect the flexibility and conformation of residues connecting the C and D helices, referred to as the CD corner, which is coupled to the configuration adopted by the distal histidine. In this study, we report the results of 30 ns MD simulations of CO-bound Ngb in the crystal. Our goal was to compare the protein dynamical behavior in the crystal with the results supplied by the previous MD simulation of CO-bound Ngb in solution and the x-ray experimental data. The results show that the different environments (crystal or solution) affect the dynamics of the heme group and of the CD corner.

Solvent electrostriction-driven peptide folding revealed by quasi-Gaussian entropy theory and molecular dynamics simulation.

A quantitative understanding of the complex relationship between microscopic structure and the thermodynamics driving peptide and protein folding is a major goal of biophysical chemistry. Here, we present a methodology comprising the use of an extended quasi-Gaussian entropy theory parametrized using molecular dynamics simulation that provides a complete description of the thermodynamics of peptide conformational states. The strategy is applied to analyze the conformational thermodynamics of MR121-GSGSW, a peptide well characterized in experimental studies. The results demonstrate that the extended state of the peptide possesses the lowest partial molar entropy. The origin of this entropy decrease is found to be in the increase of the density and orientational order of the hydration water molecules around the peptide, induced by the "unfolding". While such a reduction of the configurational entropy is usually associated with the hydrophobic effect, it is here found to be mainly due to the interaction of the solute charges with the solvent, that is, electrostriction.

Monte Carlo folding of trans-membrane helical peptides in an implicit generalized Born membrane.

An efficient Monte Carlo (MC) algorithm using concerted backbone rotations is combined with a recently developed implicit membrane model to simulate the folding of the hydrophobic transmembrane domain M2TM of the M2 protein from influenza A virus and Sarcolipin at atomic resolution. The implicit membrane environment is based on generalized Born theory and has been calibrated against experimental data. The MC sampling has previously been used to fold several small polypeptides and been shown to be equivalent to molecular dynamics (MD). In combination with a replica exchange algorithm, M2TM is found to form continuous membrane spanning helical conformations for low temperature replicas. Sarcolipin is only partially helical, in agreement with the experimental NMR structures in lipid bilayers and detergent micelles. Higher temperature replicas exhibit a rapidly decreasing helicity, in agreement with expected thermodynamic behavior. To exclude the possibility of an erroneous helical bias in the simulations, the model is tested by sampling a synthetic Alanine-rich polypeptide of known helicity. The results demonstrate there is no overstabilization of helical conformations, indicating that the implicit model captures the essential components of the native membrane environment for M2TM and Sarcolipin.

Misfolding of the amyloid beta-protein: a molecular dynamics study.

Amyloid beta-proteins spontaneously aggregate and build plaques in the brains of Alzheimer's disease patients. The polypeptide has been the subject of extensive in vitro and computational research. Still, the pathway to aggregational forms and their exact conformations remain largely unclear. Here we present an extensive molecular dynamics approach simulating the protein in various temperatures, pH conditions, and with different charge states of the N- and C-termini, thus exploring the conformational space of the protein at large. Our results show that the protein is able to sample different conformations, many of which are rich in beta structure content, and all characterized by a rapid loss of helix 1 that converts into a pi-helix, while helix 2 samples random and beta-rich structures. Moreover, a hydrophobic cluster is observed involving Val18, Phe19, Ala21, and Gly25. The results are carefully compared with recent NMR and spectroscopic data, and are in global agreement with the experimental findings.

Aggregation of small peptides studied by molecular dynamics simulations.

Peptides and proteins tend to aggregate under appropriate conditions. The amyloid fibrils that are ubiquitously found among these structures are associated with major human diseases like Alzheimer's disease, type II diabetes, and various prion diseases. Lately, it has been observed that even very short peptides like tetra and pentapeptides can form ordered amyloid structures. Here, we present aggregation studies of three such small polypeptide systems, namely, the two amyloidogenic peptides DFNKF and FF, and a control (nonamyloidogenic) one, the AGAIL. The respective aggregation process is studied by all-atom Molecular Dynamics simulations, which allow to shed light on the fine details of the association and aggregation process. Our analysis suggests that naturally aggregating systems exhibit significantly diverse overall cluster shape properties and specific intermolecular interactions. Additional analysis was also performed on the previously studied NFGAIL system.

Monte Carlo vs molecular dynamics for all-atom polypeptide folding simulations.

An efficient Monte Carlo (MC) algorithm including concerted rotations is directly compared to molecular dynamics (MD) in all-atom statistical mechanics folding simulations of small polypeptides. The previously reported algorithm "concerted rotations with flexible bond angles" (CRA) has been shown to successfully locate the native state of small polypeptides. In this study, the folding of three small polypeptides (trpzip2/H1/Trp-cage) is investigated using MC and MD, for a combined sampling time of approximately 10(11) MC configurations and 8 micros, respectively. Both methods successfully locate the experimentally determined native states of the three systems, but they do so at different speed, with 2-2.5 times faster folding of the MC runs. The comparison reveals that thermodynamic and dynamic properties can reliably be obtained by both and that results from folding simulations do not depend on the algorithm used. Similar to previous comparisons of MC and MD, it is found that one MD integration step of 2 fs corresponds to one MC scan, revealing the good sampling of MC. The simplicity and efficiency of the MC method will enable its future use in folding studies involving larger systems and the combination with replica exchange algorithms.

On the effect of a point mutation on the reactivity of CuZn superoxide dismutase: a theoretical study.

In this paper, we investigate the effects of a point mutation on the enzymatic activity of copper-zinc superoxide dismutase, which we recently studied in detail by means of a theoretical-computational procedure. Comparison of the reactivity of the initial catalytic steps in this mutant (G93A mutation far from the active site) with our previous data, reveals the beautiful mechanical-dynamical architecture of the enzyme, altered by such an apparently irrelevant mutation. Finally, our results suggest a possible atomic-molecular-based explanation for the mutant-pathology correlation, in line with the most recent experimental data.

Thermodynamic and kinetic characterization of a beta-hairpin peptide in solution: an extended phase space sampling by molecular dynamics simulations in explicit water.

The folding of the amyloidogenic H1 peptide MKHMAGAAAAGAVV taken from the syrian hamster prion protein is explored in explicit aqueous solution at 300 K using long time scale all-atom molecular dynamics simulations for a total simulation time of 1.1 mus. The system, initially modeled as an alpha-helix, preferentially adopts a beta-hairpin structure and several unfolding/refolding events are observed, yielding a very short average beta-hairpin folding time of approximately 200 ns. The long time scale accessed by our simulations and the reversibility of the folding allow to properly explore the configurational space of the peptide in solution. The free energy profile, as a function of the principal components (essential eigenvectors) of motion, describing the main conformational transitions, shows the characteristic features of a funneled landscape, with a downhill surface toward the beta-hairpin folded basin. However, the analysis of the peptide thermodynamic stability, reveals that the beta-hairpin in solution is rather unstable. These results are in good agreement with several experimental evidences, according to which the isolated H1 peptide adopts very rapidly in water beta-sheet structure, leading to amyloid fibril precipitates [Nguyen et al., Biochemistry 1995;34:4186-4192; Inouye et al., J Struct Biol 1998;122:247-255]. Moreover, in this article we also characterize the diffusion behavior in conformational space, investigating its relations with folding/unfolding conditions.

Molecular dynamics simulation of the aggregation of the core-recognition motif of the islet amyloid polypeptide in explicit water.

The formation of amyloid fibrils is associated with major human diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood. Here, we used molecular dynamics simulations to study the initial self-assembly stages of the NH2-NFGAIL-COOH peptide, the core-recognition motif of the type II diabetes associated islet amyloid polypeptide. The simulations were performed using multiple replicas of the monomers in explicit water, in a confined box starting from a random distribution of the peptides at T = 300 K and T = 340 K. At both temperatures the formation of unique clusters was observed after a few nanoseconds. Structural analysis of the clusters clearly suggested the formation of "flat" ellipsoid-shaped clusters through a preferred locally parallel alignment of the peptides. The unique assembly is facilitated by a preference for an extended conformation of the peptides and by intermolecular aromatic interactions. Taken together, our results may provide a description of the molecular recognition determinants involved in fibril formation, in terms of the atomic detailed structure of nascent aggregates. These observations may yield information on new ways to control this process for either materials development or drug design.

Properties of integral membrane protein structures: derivation of an implicit membrane potential.

Distributions of each amino acid in the trans-membrane domain were calculated as a function of the membrane normal using all currently available alpha-helical membrane protein structures with resolutions better than 4 A. The results were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans-membrane alpha-helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For alpha-helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans-membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials.

Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates.

Glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of L-glutamine amide group to the C2 carbon of 2-oxoglutarate yielding two molecules of L-glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS alpha subunit and the structure of the reduced enzyme in complex with the L-glutamine and 2-oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS alpha subunit in complex with L-methionine sulphone and 2-oxoglutarate. The present 4-ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263-271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q-loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L-methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid-body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS alpha subunit.

Dynamic effects of mutations within two loops of cytochrome c551 from Pseudomonas aeruginosa.

In this work, we investigated the structural and dynamic consequences of two substitutions, P58A and G36P, located in two different solvent-exposed loops of cytochrome c551. The results show that both mutations affect regions that are distant from the site of mutation. Here, the two loops appear to be dynamically coupled to each other, because the substitution at one site affects the structure and the dynamics of the other site. However, the substitutions at Gly-36 and Pro-58 presented substantial differences, which were related to the mechanical (rigidity and deformability) properties of the site surrounding the mutation. Although the P58A mutant conserved a significant dynamic similarity to the wild-type protein as the immediate surroundings of position 58 became more rigid, the G36P mutant, which had deformed its flexible surroundings, presented a dynamic behavior that was markedly different from that of the wild-type protein. These results suggest that perturbation of sterically isolated and flexible regions, such as solvent-exposed loops, can have strong dynamic consequences on the protein as a whole, raising the possibility that these effects could in turn affect the stability or the function of the protein.

Beta-hairpin conformation of fibrillogenic peptides: structure and alpha-beta transition mechanism revealed by molecular dynamics simulations.

Understanding the conformational transitions that trigger the aggregation and amyloidogenesis of otherwise soluble peptides at atomic resolution is of fundamental relevance for the design of effective therapeutic agents against amyloid-related disorders. In the present study the transition from ideal alpha-helical to beta-hairpin conformations is revealed by long timescale molecular dynamics simulations in explicit water solvent, for two well-known amyloidogenic peptides: the H1 peptide from prion protein and the Abeta(12-28) fragment from the Abeta(1-42) peptide responsible for Alzheimer's disease. The simulations highlight the unfolding of alpha-helices, followed by the formation of bent conformations and a final convergence to ordered in register beta-hairpin conformations. The beta-hairpins observed, despite different sequences, exhibit a common dynamic behavior and the presence of a peculiar pattern of the hydrophobic side-chains, in particular in the region of the turns. These observations hint at a possible common aggregation mechanism for the onset of different amyloid diseases and a common mechanism in the transition to the beta-hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the alpha-helical conformations induced by the presence of an organic fluorinated cosolvent. The results of MD simulation in 2,2,2-trifluoroethanol (TFE)/water mixture provide further evidence that the peptide coating effect of TFE molecules is responsible for the stabilization of the soluble helical conformation.

Kinetics of carbon monoxide migration and binding in solvated neuroglobin as revealed by molecular dynamics simulations and quantum mechanical calculations.

Neuroglobin (Ngb) is a globular protein that reversibly binds small ligands at the six coordination position of the heme. With respect to other globins similar to myoglobin, Ngb displays some peculiarities as the topological reorganization of the internal cavities coupled to the sliding of the heme, or the binding of the endogenous distal histidine to the heme in the absence of an exogenous ligand. In this Article, by using multiple (independent) molecular dynamics trajectories (about 500 ns in total), the migration pathways of photolized carbon monoxide (CO) within solvated Ngb were analyzed, and a quantitative description of CO migration and corresponding kinetics was obtained. MD results, combined with quantum mechanical calculations on the CO-heme binding-unbinding reaction step in Ngb, allowed construction of a quantitative model representing the relevant steps of CO migration and rebinding.