The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider.

Gomesin is the first peptide isolated from spider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four cysteines forming two disulfide linkages. The solution structure of gomesin has been determined using proton two-dimensional NMR (2D-NMR) and restrained molecular dynamics calculations. The global fold of gomesin consists in a well-resolved two-stranded antiparallel betasheet connected by a noncanonical betaturn. A comparison between the structures of gomesin and protegrin-1 from porcine and androctonin from scorpion outlines several common features in the distribution of hydrophobic and hydrophilic residues. The N- and C-termini, the betaturn and one face of the betasheet are hydrophilic, but the hydrophobicity of the other face depends on the peptide. The similarities suggest that the molecules interact with membranes in an analogous manner. The importance of the intramolecular disulfide bridges in the biological activity of gomesin is being investigated.     

Solution conformation of a cyclic neurokinin antagonist: A nmr and molecular dynamics study

The solution structure of a hexapeptide, cyclo(Gln-Trp-Phe-Gly-Leu-Met), which is a selective NK-2 antagonist, has been studied by a combination of two-dimensional nmr and molecular dynamics (MD) techniques. The simulation based on nmr and MD data resulted in the convergence to a family of structures. Free molecular dynamics for 50 ps in the presence of DMSO solvent molecules shows that the structure is energetically stable. One intramolecular hydrogen bond between the amide proton of Gin and the carbonyl oxygen of Gly was revealed. This result is consistent with the results from the measurement of the temperature coefficient of the amide protons. The extent of intermolecular hydrogen bonding between the amide protons of the peptide and DMSO was also revealed by the free MD simulation. The resulting structure of the cyclic peptide contains a variation type I′ β-turn in the Gly-Leu-Met-Gln segment. Comparison of the structure of this peptide with that of other NK-2 antagonist cyclic hexapeptides was made, and the activity of cyclic antagonists appears to be inversely related to the conformational rigidity of the cyclic peptides. © 1994 John Wiley & Sons, Inc.     

Theoretical studies of the structure and molecular dynamics of a peptide crystal

Energy minimizations and molecular dynamics simulations have been performed on the cyclic peptide cyclo-(Ala-Pro-D-Phe)2 in both the isolated and crystal states. The results of these calculations have been analyzed, both to investigate our ability to reproduce experimental data (structure and vibrational and NMR spectra) and to investigate the effects of environment on the energy, structure, and dynamics of peptides. Comparison of the minimized and time-averaged crystal systems with the experimental peptide structure shows that the calculations have closely reproduced the experimental structure. Molecular dynamics of the isolated molecule has led to a new conformation, which is approximately equal to 8.5 kcal/mol more stable than the conformation that exists in the crystal, the latter conformation being stabilized by intermolecular (packing) forces. This illustrates the considerable effect that environment can have on the conformation of peptides. The crystal environment has also been shown to significantly reduce the dynamic conformational fluctuations seen for the isolated molecule. The behavior of the peptide during the isolated simulation also supports the experimental NMR observation of a symmetric structure that differs from the asymmetric, instantaneous structures which characterize the molecule during the dynamics. Calculations of vibrational frequencies of the peptide in the crystal and isolated states show the expected shifts in bond-stretching frequencies due to intermolecular interactions. Finally, we have calculated NMR coupling constants from the dynamics simulation of the isolated peptide and have compared these with the experimental values. This has led to a possible reinterpretation of the experimental data.     

Cooperative folding mechanism of a beta-hairpin peptide studied by a multicanonical replica-exchange molecular dynamics simulation

G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific beta-hairpin within 6 micros. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a multicanonical replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like beta-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high phi-values of the turn region observed by experiment.     

Conformational flexibility of the MHC class I alpha1-alpha2 domain in peptide bound and free states: a molecular dynamics simulation study

Major histocompatibility complex class I proteins play a key role in the recognition and presentation of peptide antigens to the host immune system. The structure of various major histocompatibility complex class I proteins has been determined experimentally in complex with several antigenic peptides. However, the structure in the unbound (empty) form is not known. To study the conformational dynamics of the empty major histocompatibility complex class I molecule comparative molecular dynamics simulations have been performed starting from the crystal structure of a peptide bound class I peptide-binding domain in the presence and absence of a peptide ligand. Simulations including the bound peptide stayed close to the experimental start structure at both simulation temperatures (300 and 355 K) during the entire simulation of 26 ns. Several independent simulations in the absence of peptide indicate that the empty domain may not adopt a single defined conformation but is conformationally significantly more heterogeneous in particular within the alpha-helices that flank the peptide binding cleft. The calculated conformational dynamics along the protein chain correlate well with available spectroscopic data and with the observed site-specific sensitivity of the empty class I protein to proteolytic digestion. During the simulations at 300 K the binding region for the peptide N-terminus stayed close to the conformation in the bound state, whereas the anchor region for the C-terminus showed significantly larger conformational fluctuations. This included a segment at the beginning of the second alpha-helix in the domain that is likely to be involved in the interaction with the chaperone protein tapasin during the peptide-loading process. The simulation studies further indicate that peptide binding at the C- and N-terminus may follow different mechanisms that involve different degrees of induced conformational changes in the peptide-binding domain. In particular binding of the peptide C-terminus may require conformational stabilization by chaperone proteins during peptide loading.     

Molecular dynamics simulations of a highly charged peptide from an SH3 domain: possible sequence-function relationship

A seven-residue peptide that is highly conserved in SH3 domains despite being far from the active site has been shown by NMR to be stable in solution. This peptide, biologically important because it is a likely folding nucleus for SH3 domains, provides a challenging subject for molecular dynamics because it is highly charged. We present stable, 10-ns simulations of both the native-like diverging turn structure and a helical model. Free energies of these two conformations, estimated through MM-PBSA analysis using several force fields, suggest a comparable free energy (DeltaDeltaG < or =6 kcal/mol) for native and helix conformations. NOE intensities calculated from the native trajectory reproduce experimental data quite well, suggesting that the conformations sampled by the trajectory reasonably represent those observed in the NMR experiment. The molecular dynamics results, as well as sequence analysis of a diverse 690-member family of SH3 domain proteins, suggest that the presence of two elements is essential for formation of the diverging turn structure: a pair of residues with low helical propensity in the turn region and, as previously recognized, two hydrophobic residues to close the end of the diverging turn. Thus, these two sequence features may form the structural basis for the function of this peptide as a folding nucleus in this family of proteins.     

Rotamer interconversion and its influence on the fluorescence decay of tyrosine: a molecular dynamics study.

To test the hypothesis of charge-transfer quenching between an electrophile in the alanyl sidechain (carbonyl carbon or protonated amino group) and the excited aromatic phenol-subunit, which leads to a bi-exponential fluorescence decay of tyrosine in acidic aqueous solution, we investigated the dynamics of this amino acid and of the peptide Gly-Tyr-Gly in vacuo and in water with classical molecular dynamics (MD) and with stochastic dynamics (SD) computer simulation. The proposed low-frequency of interconversions between sidechain rotamers on a fluorescence time-scale could not be confirmed. Instead, frequent transitions for both, chi 1 and chi 2, dihedrals were observed. Simulating a low pH situation (protonated carboxylate group) did not significantly affect the transition frequency. Rotamer interconversions in the peptide Gly-Tyr-Gly, though significantly less, were also observed although the fluorescence decay of this compound could be described by a uni-modal lifetime distribution centered at 0.8 ns. The results obtained from simulations in vacuo and in solution were critically compared with those of stochastic simulations. We found the stochastic simulation in a better agreement to full MD (water explicitly included), which is highly time consuming, whereas the in vacuo simulations clearly deviated from both. We conclude from our results that, since the rotamers do frequently interconvert within the fluorescence lifetime of tyrosine, their contribution to the non-exponential fluorescence decay should be negligible.     

Conformational transitions in the nootropic peptide semax (MEHFPGP) and its N-terminal modifications

We use molecular dynamics simulation to examine the conformational possibilities in solution for the peptide MEHFPGP (Semax) representing the minimal nootropic fragment of MSH, and its versions with N-terminal substitutions of K, G, or R for M. We discuss the possible relationship between molecule structure and physiological activity, considering the influence of Coulomb interactions on the dynamics and the putative stabilization of a certain peptide conformation at pH < 6.     

Direct observation of protein folding, aggregation, and a prion-like conformational conversion

Protein conformational transition from alpha-helices to beta-sheets precedes aggregation of proteins implicated in many diseases, including Alzheimer and prion diseases. Direct characterization of such transitions is often hindered by the complicated nature of the interaction network among amino acids. A recently engineered small protein-like peptide with a simple amino acid composition features a temperature-driven alpha-helix to beta-sheet conformational change. Here we studied the conformational transition of this peptide by molecular dynamics simulations. We observed a critical temperature, below which the peptide folds into an alpha-helical coiled-coil state and above which the peptide misfolds into beta-rich structures with a high propensity to aggregate. The structures adopted by this peptide during low temperature simulations have a backbone root mean square deviation less than 2 A from the crystal structure. At high temperatures, this peptide adopts an amyloid-like structure, which is mainly composed of coiled anti-parallel beta-sheets with the cross-beta-signature of amyloid fibrils. Most strikingly, we observed conformational conversions in which an alpha-helix is converted into a beta-strand by proximate stable beta-sheets with exposed hydrophobic surfaces and unsaturated hydrogen bonds. Our study suggested a possible generic molecular mechanism of the template-mediated aggregation process, originally proposed by Prusiner (Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13363-13383) to account for prion infectivity.     

Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family

We have purified a small size antimicrobial peptide, named gomesin, from the hemocytes of the unchallenged tarantula spider Acanthoscurria gomesiana. Gomesin has a molecular mass of 2270.4 Da, with 18 amino acids, including a pyroglutamic acid as the N terminus, a C-terminal arginine alpha-amide, and four cysteine residues forming two disulfide bridges. This peptide shows marked sequence similarities to antimicrobial peptides from other arthropods such as tachyplesin and polyphemusin from horseshoe crabs and androctonin from scorpions. Interestingly, it also shows sequence similarities to protegrins, antimicrobial peptides from porcine leukocytes. Gomesin strongly affects bacterial growth, as well as the development of filamentous fungi and yeast. In addition, we showed that gomesin affects the viability of the parasite Leishmania amazonensis.     

Local unfolding of Cu, Zn superoxide dismutase monomer determines the morphology of fibrillar aggregates

Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Conformation of a peptide encompassing the proton translocation channel of vacuolar H(+)-ATPase

The structural properties of a crucial transmembrane helix for proton translocation in vacuolar ATPase are studied using double site-directed spin-labeling combined with electron spin resonance (ESR) (or electron paramagnetic resonance) and circular dichroism spectroscopy in sodium dodecyl sulfate micelles. For this purpose, we use a synthetic peptide derived from transmembrane helix 7 of subunit a from the yeast Saccharomyces cerevisiae vacuolar proton-translocating ATPase that contains two natural cysteine residues suitable for spin-labeling. The interspin distance is calculated using a second-moment analysis of the methanethiosulfonate spin-label ESR spectra at 150 K. Molecular dynamics simulation is used to study the effect of the side-chain dynamics and backbone dynamics on the interspin distance. Based on the combined results from ESR, circular dichroism, and molecular dynamics simulation we conclude that the peptide forms a dynamic alpha-helix. We discuss this finding in the light of current models for proton translocation. A novel role for a buried charged residue (H729) is proposed.     

Characterizing a partially ordered miniprotein through folding molecular dynamics simulations: Comparison with the experimental data

The villin headpiece helical subdomain (HP36) is one of the best known model systems for computational studies of fast‐folding all‐α miniproteins. HP21 is a peptide fragment—derived from HP36—comprising only the first and second helices of the full domain. Experimental studies showed that although HP21 is mostly unfolded in solution, it does maintain some persistent native‐like structure as indicated by the analysis of NMR‐derived chemical shifts. Here we compare the experimental data for HP21 with the results obtained from a 15‐μs long folding molecular dynamics simulation performed in explicit water and with full electrostatics. We find that the simulation is in good agreement with the experiment and faithfully reproduces the major experimental findings, namely that (a) HP21 is disordered in solution with <10% of the trajectory corresponding to transiently stable structures, (b) the most highly populated conformer is a native‐like structure with an RMSD from the corresponding portion of the HP36 crystal structure of <1 Å, (c) the simulation‐derived chemical shifts—over the whole length of the trajectory—are in reasonable agreement with the experiment giving reduced χ² values of 1.6, 1.4, and 0.8 for the Δδ¹³C^α, Δδ¹³CO, and Δδ¹³C^β secondary shifts, respectively (becoming 0.8, 0.7, and 0.3 when only the major peptide conformer is considered), and finally, (d) the secondary structure propensity scores are in very good agreement with the experiment and clearly indicate the higher stability of the first helix. We conclude that folding molecular dynamics simulations can be a useful tool for the structural characterization of even marginally stable peptides.     

Structural and dynamical studies of Humanin in water and TFE/water mixture: a molecular dynamics simulation

The structural and dynamical properties of Humanin, a small peptide with neuroprotective activity against the insults of the Alzheimer's disease-related genes and the neurotoxic amyloid peptide, are studied in two different environments by molecular dynamics simulation. In this study, we have performed comparative molecular dynamics simulations in the absence and in the presence of TFE. The resulting trajectories were analyzed in terms of structural and dynamical properties of peptide and compared to the available NMR data. In water humanin is observed to partly unfold. The peptide is readily stabilized in an ordered helical conformation in the TFE/water mixture. Our simulations show that the peptide is flexible with definite turn point in its structure in water environment. It is free to interact with receptors that mediate its action in polar environment. Humanin may also find an alpha helix structure necessary for passage through biomembranes and/or specific interactions.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Structural investigation of syringomycin-E using molecular dynamics simulation and NMR

Syringomycin-E (SR-E) is a cyclic lipodepsinonapeptide produced by certain strains of the bacterium Pseudomonas syringae pv. syringae. It shows inhibitory effects against many fungal species, including human pathogens. Its primary biological target is the plasma membrane, where it forms channels comprised of at least six SR-E molecules. The high-resolution structure of SR-E and the structure of the channels are currently not known. In this paper, we investigate in atomic detail the molecular features of SR-E in water by NMR and in water and octane by molecular dynamics simulation (MD). We built a model of the peptide and examined its structure in water and octane in 200 ns MD simulations both with and without distance restraints derived from NMR NOE data. The resulting trajectories show good agreement with the measured NOEs and circular dichroism data from the literature and provide atomistic models of SR-E that are an important step toward a better understanding of the antifungal and antibacterial activity of this peptide.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Structural aspects of Ca2+ binding by acyclic peptides: low-energy conformational domains and molecular dynamics of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide.

We have applied random-search, energy minimization and molecular dynamics simulations to investigate the structural aspects of the interaction of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide with Ca2+. Spectral data on related peptides had suggested that the beta-turn conformation might be a prerequisite for the binding of cation ion by such short linear peptides. In order to relate the conformational characteristics with the Ca(2+)-binding affinities of these peptides, the molecular events involved in cation binding need to be understood. We have addressed this problem in this study by using a systematic approach that involved the following steps. First, a random search technique was used to generate a large population of conformers for the free peptide in the absence of Ca2+. Next, the energies of these conformers were computed. Conformations with energies within 4 kcal/mol of the global minimum were analysed and found to fall into four main groups characterized by the presence of different types of hydrogen-bonded structures including single and consecutive beta-turns. The energies for interconversion of conformers from one group to another were computed and found to be relatively small (< 10 kcal/mol). Finally, molecular dynamics of the peptide at 300K in the presence of Ca2+ were used to simulate the cation binding process. Starting points for these simulations were generated by placing the ion in the vicinity of two molecules of the peptide. The simulation results showed that the conformers with two consecutive beta-turns led to the formation of a stable 2:1 (peptide:Ca2+) sandwich complex in agreement with earlier experimental observations on similar linear peptides. While the starting conformation of the peptide in the consecutive beta-turn structure allowed for the proper orientation of three carbonyl oxygen atoms for chelation to the metal ion, the dynamics of complex formation rearranged the peptide structure substantially, leading to the formation of an 8-coordinated Ca2+ complex in a dodecahedral spatial arrangement. Thus, based on the energetics of the structures and processes involved, the present study demonstrates that: a) peptide-Ca2+ complex formation is initiated by conformers adopting consecutive beta-turn structures which subsequently go over to a significantly different conformation found in the complex; and, b) The facile interconversion between the low-energy conformers in the different groups would help shift the equilibrium population towards the consecutive beta-turn structure during the complex formation.     

Effect of hexafluoroisopropanol alcohol on the structure of melittin: a molecular dynamics simulation study

The molecular mechanism by which HFIP stabilizes the alpha-helical structure of peptides is not well understood. In the present study, we use melittin as a model to gain insight into the details of the atomistic interactions of HFIP with the peptide. We have performed extensive comparative molecular dynamics simulations (up to 100 nsec) in the absence and in the presence of HFIP. In agreement with recent NMR experiments, the simulations show rapid loss of tertiary structure in water at pH 2 but much higher helicity in 35% HFIP. The MD simulations also indicate that melittin adopts a highly dynamic global structure in 35% HFIP solution with two alpha-helical segments sampling a wide range of angular orientations. The analysis of the HFIP distribution shows the tendency of HFIP to aggregate around the peptide, increasing the local cosolvent concentration to more than two times that in the bulk concentration. The correlation of local peptide structure with HFIP coating suggests that displacement of water at the peptide surface is the main contribution of HFIP in stabilizing the secondary structure of melittin. Finally, a stabilizing effect promoted by the presence of counter-ions was also observed in the simulations.