Conformational behavior of temporin A and temporin L in aqueous solution: a computational/experimental study.

Molecular dynamics (MD) simulations and circular dichroism (CD) experiments were carried out on aqueous temporin A and L, two short peptides belonging to an interesting class of natural substances known to be active mainly against Gram-positive/negative bacteria and fungi. Experimental results indicate the higher propensity of temporin L, with respect to temporin A, in forming alpha-helical structures. These results were revisited by long-timescale MD simulations, in which their alpha-helical propensity was investigated in the absence of trifluoroethanol. Results clearly show the higher stability of alpha-helix conformations in temporin L; moreover, an interestingly strong mechanical analogy emerges since both temporins show the same residue interval (from 7 to 10) as the most energetically accessible for alpha-helix formation. Such studies provide some intriguing structural and mechanical evidence that may help in better understanding and rationalizing the conformational behaviour of temporins in water solution and, ultimately, the inner principles of their microbial targets selectivity and mechanism of action at the level of cell membranes.

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.

Conformations in solution of the fuscopeptins. Phytotoxic metabolites of Pseudomonas fuscovaginae.

Fuscopeptins are phytotoxic amphiphilic lipodepsipeptides containing 19 amino acid residues. They are produced by the plant pathogenic bacterium Pseudomonas fuscovaginae in two forms, A and B, which differ only in the number of methylene groups in the fatty acid chain. Their covalent structure and biological properties have been reported previously. CD and NMR spectroscopy investigations in solution revealed the absence of identifiable elements of secondary and tertiary structure for these molecules. Fuscopeptin B appears to be completely unstructured in aqueous solution, and has a large molecular flexibility. A dramatic conformational change was observed upon addition of trifluoroethanol. This study reports the complete interpretation of the two-dimensional NMR spectra and the NOE results obtained for fuscopeptin B in water/trifluoroethanol solutions; the signals relative to the peptidic moiety are identical to those observed for fuscopeptin A. The results of this investigation were used to determine the solution structure of fuscopeptin B by computer simulations applying distance geometry and simulated annealing procedures. In water/trifluoroethanol solutions the peptidic region appears to have a partly helical structure. The lactonic ring assumes defined conformations very similar to those already reported for other lipodepsipeptides. The structure for fuscopeptin B in solution is also valid for fuscopeptin A because of the negligible structural difference between the two metabolites.

On the convergence of the conformational coordinates basis set obtained by the essential dynamics analysis of proteins' molecular dynamics simulations.

In this article we present a quantitative evaluation of the convergence of the conformational coordinates of proteins, obtained by the Essential Dynamics method. Using a detailed analysis of long molecular dynamics trajectories in combination with a statistical assessment of the significance of the measured convergence, we obtained that simulations of a few hundreds of picoseconds are in general sufficient to provide a stable and statistically reliable definition of the essential and near constraints subspaces, at least within the nanoseconds time range. Proteins 1999;36:419-424.

Effects of core-packing on the structure, function, and mechanics of a four-helix-bundle protein ROP.

The effects of core-packing on the structure, function and mechanics of the RNA-binding 4-helix-bundle Rop have been studied by molecular dynamics simulations. The structural, dynamical and geometrical properties of the Rop homodimer, (formed by the antiparallel juxtaposition of two helix-turn-helix motifs), have been compared with those of three protein variants described by Munson et al. (Protein Sci, 5:1584-1593, 1996), where the core of the native protein has been systematically repacked using a two-amino acid alphabet: Ala(2)Leu(2)-8, Ala(2)Leu(2)-8-rev, and Leu(2)Ala(2)-8. The results showed that it was possible to readily distinguish the inactive protein Leu(2)Ala(2)-8 from the other functionally active systems based on tertiary and quaternary structure criteria. Structural properties such as native secondary structure content did not correlate with biological activity. Biological activity was related in part to the relative arrangement of the residues within the binding site. But, more global aspects, related to the overall topology of the helical bundle, accounted for the small functional differences between Ala(2)Leu(2)-8 and Ala(2)Leu(2)-8-rev. Mechanically, the 4-helix-bundle absorbed core mutations by altering the local structure at the sequence termini and in the turns that join the two helices of each monomer, and by changing the overall orientation and separation of the extremely rigid helices. Proteins 1999;36:436-446.

Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation.

In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al. (Di Nola et al., Proteins 1994;19:174-182). The method allows a fast exploration of the receptor surface, using a high temperature of the center of mass translational motion, while the ligand internal motions, the solvent, and the receptor are simulated at room temperature. In addition, the method allows a fast center of mass motion of the ligand, even in solution. The dampening effect of the solvent can be overcome by applying different weights to the interactions between system subsets (solvent, receptor, and ligand). Specific ligand-receptor distances have been used to compare the results of the simulations with the crystal structure. The method is applied, as a test system, to the docking of the phosphocholine to the immunoglobulin McPC603. The results show the similarity of structure between the complex in solution and in the crystal.

A kinetic model for the internal motions of proteins: diffusion between multiple harmonic wells.

The dynamics of collective protein motions derived from Molecular Dynamics simulations have been studied for two small model proteins: initiation factor I and the B1 domain of Protein G. First, we compared the structural fluctuations, obtained by local harmonic approximations in different energy minima, with the ones revealed by large scale molecular dynamics (MD) simulations. It was found that a limited set of harmonic wells can be used to approximate the configurational fluctuations of these proteins, although any single harmonic approximation cannot properly describe their dynamics. Subsequently, the kinetics of the main (essential) collective protein motions were characterized. A dual-diffusion behavior was observed in which a fast type of diffusion switches to a much slower type in a typical time of about 1-3 ps. From these results, the large backbone conformational fluctuations of a protein may be considered as "hopping" between multiple harmonic wells on a basically flat free energy surface.

Mechanics and dynamics of B1 domain of protein G: role of packing and surface hydrophobic residues.

The structural organization of the B1 domain of streptococcal protein G (PGA) has been probed using molecular dynamics simulations, with a particular emphasis on the role of the solvent exposed Ile6 residue. In addition to the native protein (WT-PGA), three single-mutants (I6G-PGA, I6F-PGA, and I6T-PGA), one double-mutant (I6T,T53G-PGA), and three isolated peptide fragments (corresponding to the helix and the two beta-hairpins) were studied in the presence of explicit water molecules. Comparative analysis of the various systems showed that the level of perturbation was directly related to the hydrophobicity and the size of the side chain of residue 6, the internal rigidity of the proteins decreasing in the order I6T-PGA > I6G-PGA > WT-PGA > I6F-PGA. The results emphasized the importance of residue 6 in controlling both the integrity of the sheet's surface and the orientation of the helix in relation to the sheet by modulation of surface/core interactions. The effects of mutations were delocalized across the structure, and glycine residues, in particular, absorbed most of the introduced strain. A qualitative structural decomposition of the native fold into elementary building-blocks was achieved using principal component analysis and mechanical response matrices. Within this framework, internal motions of the protein were described as coordinated articulations of these structural units, mutations affecting mostly the amplitude of the motions rather than the structure/location of the building-blocks. Analysis of the isolated peptidic fragments suggested that packing did not play a determinant role in defining the elementary building-blocks, but that chain topology was mostly responsible.

Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data.

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.

The essential dynamics of Cu, Zn superoxide dismutase: suggestion of intersubunit communication.

A 300-ps molecular dynamics simulation of the whole Cu, Zn superoxide dismutase dimer has been carried out in water, and the trajectory has been analyzed by the essential dynamics method. The results indicate that the motion is defined by few preferred directions identified by the first four to six eigenvectors and that the motion of the two monomers at each instant is not symmetrical. The vectors symmetrical to the eigenvectors are significantly sampled, suggesting that, on average, the motions of the two subunits will exchange. Large intra- and intersubunit motions involving different subdomains of the protein are observed. A mechanical coupling between the two subunits is also suggested, because displacements of the loops surrounding the active site in one monomer are correlated with the motion of parts of the second toward the intersubunit interface.

Conformational study of [Met5]enkephalin-Arg-Phe in the presence of phosphatidylserine vesicles.

The interaction of [Met5]enkephalin-Arg.Phe with phosphatidylserine (PtdSer) was studied by circular dichroism (CD), two-dimensional nuclear magnetic resonance spectroscopy, hybrid distance geometry simulated annealing (DG-SA) and molecular dynamics (MD) calculations. The very low solubility of [Met5]enkephalin-Arg-Phe and the instability of the solution containing PtdSer vesicles at low pH values did not allow us to observe the amide proton resonances in the usual two-dimensional NMR work. NOESY cross-peaks of protons of side chains from two-dimensional NMR were converted into distances which were used as restraints for modelling with DG-SA and MD. Our results indicate that, in aqueous solutions at pH 7.68 [Met5]enkephalin-Arg-Phe exists in the absence of PtdSer as a random distribution of conformers, whereas in the presence of PtdSer it adopts conformations containing a common orientation of the bonds of C alpha 2, C alpha 3, C alpha 4, and C alpha 5, although different orientations of the peptide planes are consistent with the results. Two of the reported conformers from MD simulations are characterized by the presence of a 2<--4 gamma and inverse gamma turns centered on Gly3. A gradual decline of order was observed when moving from the central moiety of the peptide to both the N-terminus and C-terminus. Finally, the DG-SA and MD calculations resulted in a structure such that the orientation of the Phe4 and Met5 side chains favours hydrophobic interactions with the apolar portion of the PtdSer vesicle to form a hydrophobic cluster. These data support the hypothesis of a role of lipids to modify the conformation of [Met5]enkephalin-Arg-Phe to permit the interactions with the receptor site.

Solution conformation of the Pseudomonas syringae pv. syringae phytotoxic lipodepsipeptide syringopeptin 25-A. Two-dimensional NMR, distance geometry and molecular dynamics.

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The conformation of syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-2-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three-dimensional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported.

Molecular dynamics simulation of the docking of substrates to proteins.

A simple method is described to perform docking of substrates to proteins or probes to receptor molecules by a modification of molecular dynamics simulations. The method consists of a separation of the center-of-mass motion of the substrate from its internal and rotational motions, and a separate coupling to different thermal baths for both types of motion of the substrate and for the motion of the receptor. Thus the temperatures and the time constants of coupling to the baths can be arbitrarily varied for these three types of motion, allowing either a frozen or a flexible receptor and allowing control of search rate without disturbance of internal structure. In addition, an extra repulsive term between substrate and protein was applied to smooth the interaction. The method was applied to a model substrate docking onto a model surface, and to the docking of phosphocholine onto immunoglobulin McPC603, in both cases with a frozen receptor. Using translational temperatures of the substrate in the range of 1300-1700 K and room temperature for the internal degrees of freedom of the substrate, an efficient nontrapping exploratory search ("helicopter view") is obtained which visits the correct binding sites. Low energy conformations can then be further investigated by separate search or by dynamic simulated annealing. In both cases the correct minima were identified. The possibility to work with flexible receptors is discussed.

Solution conformation of a pectic acid fragment by 1H-NMR and molecular dynamics.

1H-NMR and molecular dynamics simulations in vacuo and in water of (1-->4)-alpha-D-galacturono-disaccharide were performed. The results of the molecular dynamics simulations showed that the molecule fluctuates between two conformations characterized by different values of torsion angles around the glycosidic linkage and two different intramolecular hydrogen bonds. When these conformations are extrapolated to a regular polymeric structure, they generate pectic acid compatible with a 2(1)- or a right-handed 3(1)-helix.

1H and 23Na NMR relaxation times study of pectin solutions and gels.

1H and 23Na longitudinal and transverse relaxation times have been measured to examine the concentration dependence of the dynamic behaviour of pectin solutions and gels. T1 and T2 relaxation times were measured in HDE pectin solutions and gels prepared with different cosolutes. A lowering of T1 and T2 values was observed in relation to a better efficiency of the intermolecular forces between polymer molecules and/or the formation of more extended junction zones. The T1/T2 ratio was also exploited indicating that even bulk water experiences anisotropic motions due to the gel formation. A correspondence of NMR results with previous reported data of rheological measurements of pectin gels prepared with the same cosolutes was found.

NMR study of seed hydration: effect of pH and ionic strength on water uptake of soaked cowpeas.

A NMR method based on the analysis of the transverse magnetization decay curve of water protons, was employed to study the hydration process of Ife Brown cowpeas. In order to investigate the role of pH and Ionic Strength (IS) on the hydration process the beans were soaked in solutions at different IS and at different pH-constant IS. The kinetic constant for the hydration process and the water-holding capacity of the seeds in different conditions were measured.

Synthesis, conformation, and activity of HCO-Met-delta Z Leu-Phe-OMe, an active analogue of chemotactic N-formyltripeptides.

In order to induce a beta-turn conformation into the chemotactic linear tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the new analogue N-formyl-L-methionyl-delta Z leucyl-L-phenylalanine methyl ester [delta Z Leu]2fMLP-OMe (1) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-alpha,beta-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1. The x-ray analysis shows that 1 adopts in the crystal a type II beta-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. 1H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl3 solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by fMLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity.

NMR study of seed hydration: the role of the seed anatomical structures in water uptake of soaked cowpeas.

A NMR method based on the analysis of the transverse magnetization decay curve of water protons, was employed to study the hydration process of commercial cowpeas. In order to investigate the role of the different anatomical parts of the seeds, hilum and micropyle, or alternatively seed coat, were inhibited by a water resistant epoxy resin. The kinetic constant for the hydration of the different sets of beans was measured.

NMR study of seed hydration with deuterated water: dependence of proton signals on hydration level.

Proton NMR signals in seeds are shown to depend on hydration level. In fact at low water amount, as it occurs in many native seeds, protons can have a restricted mobility and are not detectable. A NMR method for measuring the dependence of proton signals on hydration is reported. The method also allows the separation of the contributions of water and non-water protons in a low-resolution NMR experiment. It is based on successive hydrations (with deuterated water) - desiccation steps and on the analysis of the transverse magnetization decay curves.