Molecular-dynamics simulations of [Met5]- and [D-Ala2,Met5]-enkephalins. Biological implication of monomeric folded and dimeric unfolded conformations.

To investigate the biologically active conformation of enkephalin, molecular-dynamics simulations were applied to [Met5]- and [D-Ala2,Met5]-enkephalins. The dynamic trajectory of monomeric extended [Met5]-enkephalin was analysed in terms of relative mobility between respective torsions of backbone chain. After 10 ps of the dynamics simulation, the conformational transition was converged into a stationary state among the beta-bend folded forms, where they are stabilized by several intramolecular hydrogen-bond formations. Similar conformational transition was also observed in the dynamics simulation of [D-Ala2,Met5]enkephalin, which is a more mu-receptor-specific peptide than [Met5]enkephalin. The geometrical correspondence between the monomeric enkephalin conformation in the stationary state and morphine molecule (a mu-specific rigid opiate) was surveyed by virtue of the triangular substructures generated by choosing three functional atoms in each molecule, and good resemblances were observed. On the other hand, the dynamics simulation of the antiparallel extended [Met5]enkephalin dimer showed a trajectory different from that of the monomeric one. Two intermolecular hydrogen bonds at Tyr1 (NH3+)...Met5(CO2-) end residues were held throughout the 100 ps simulation, the dimeric structure being consequently kept. The conformational transition of the backbone chains from the antiparallel extended form to the twisted one took place via an intermediate state. Many conformations revealed during the dynamics simulation showed that the relative orientations of each two Tyr1, Gly3, Phe4 and Met5 residues in the dimer are nearly related by a pseudo-C2-symmetry respectively, and both halves of the dimer structure could be further fitted to the monomeric folded enkephalin conformation. The monomeric and dimeric conformations of enkephalin at their stationary states are discussed in relation to the substrate-specificity for mu- and delta-opioid receptors.     

Conformations of dehydrophenylalanine containing peptides. Nuclear Overhauser effect study of two acyclic tetrapeptides

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (delta Z-Phe) at position 2 or 3, Boc-Leu-Ala-delta Z-Phe-Leu-OMe (1) and Boc-Leu-delta Z-Phe-Ala-Leu-OMe (2), have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r. spectroscopy. In peptide 1 the Leu(4) NH group appears to be partially shielded from solvent, while in peptide 2 both Ala(3) and Leu(4) NH groups show limited solvent accessibility. Extensive difference nuclear Overhauser effect (n.O.e.) studies establish the occurrence of several diagnostic inter-residue n.O.e.s (Ci alpha H----Ni+1H and NiH----Ni+1H) between backbone protons. The simultaneous observation of "mutually exclusive" n.O.e.s suggests the presence of multiple solution conformations for both peptides. In peptide 1 the n.O.e. data are consistent with a dynamic equilibrium between an -Ala-delta Z-Phe- Type II beta-turn structure and a second species with delta Z-Phe adopting a partially extended conformation with psi values of +/- 100 degrees to +/- 150 degrees. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive beta-turn structure for the -Leu-delta Z-Phe-Ala- segment and an almost completely extended conformation.     

Flexibility of bovine pancreatic trypsin inhibitor

The native conformation of a protein may be expressed in terms of the dihedral angles, phi's and psi's for the backbone, and kappa's for the side chains, for a given geometry (bond lengths and bond angles). We have developed a method to obtain the dihedral angles for a low-energy structure of a protein, starting with the X-ray structure; it is applied here to examine the degree of flexibility of bovine pancreatic trypsin inhibitor. Minimization of the total energy of the inhibitor (including nonbonded, electrostatic, torsional, hydrogen bonding, and disulfide loop energies) yields a conformation having a total energy of -221 kcal/mol and a root mean square deviation between all atoms of the computed and experimental structures of 0.63 A. The optimal conformation is not unique, however, there being at least two other conformations of low-energy (-222 and -220 kcal/mol), which resemble the experimental one (root mean square deviations of 0.66 and 0.64 A, respectively). These three conformations are located in different positions in phi, psi space, i.e., with a total deviation of 81 degrees, 100 degrees and 55 degrees from each other (with a root mean square deviation of several degrees per dihedral angle from each other). The nonbonded energies of the backbones, calculated along lines in phi, psi space connecting these three conformations, are all negative, without any intervening energy barriers (on an energy contour map in the phi, psi plane). Side chains were attached at several representative positions in this plane, and the total energy was minimized by varying the kappa's. The energies were of approximately the same magnitude as the previous ones, indicating that the conformation of low energy is flexible to some extent in a restricted region of phi, psi space. Interestingly, the difference delta phi i+1 in phi i+1 for the (i + 1)th residue from one conformation to another is approximately the same as -delta psi i for the ith residue; i.e., the plane of the peptide group between the ith and (i + 1)th residues re-orient without significant changes in the positions of the other atoms. The flexibility of the orientations of the planes of the peptide groups is probably coupled in a cooperative manner to the flexibility of the positions of the backbone and side-chain atoms.     

Stacking-unstacking of the dinucleoside monophosphate guanylyl-3'',5''-uridine studied with molecular dynamics.

Molecular dynamics simulations were carried out on two conformations of the dinucleoside monophosphate guanylyl-3',5'-uridine (GpU) in aqueous solution with one sodium counterion. One stacked conformation and one with the C3'-O3'-P-O5' backbone torsion angle twisted 180 degrees to create an unstacked conformation. We observed a relatively stable behavior of the stacked conformation, which remained stacked throughout the simulation, whereas the unstacked conformation showed major changes in the backbone torsion and glycosidic angles. During the simulation the unstacked conformation transformed into a more stacked form and then back again to an unstacked one. The calculated correlation times for rotational diffusion from the molecular dynamics simulations are in agreement with fluorescence anisotropy and nuclear magnetic resonance data. As expected, the correlation times for rotational diffusion of the unstacked conformation were observed to be longer than for the stacked conformation. The 2'OH group may contribute in stabilizing the stacked conformation, where the O2'-H...O4' hydrogen bond occurred in 82.7% of the simulation.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Hydrophobic Core Formation and Dehydration in Protein Folding Studied by Generalized-Ensemble Simulations

Despite its small size, chicken villin headpiece subdomain HP36 folds into the native structure with a stable hydrophobic core within several microseconds. How such a small protein keeps up its conformational stability and fast folding in solution is an important issue for understanding molecular mechanisms of protein folding. In this study, we performed multicanonical replica-exchange simulations of HP36 in explicit water, starting from a fully extended conformation. We observed at least five events of HP36 folding into nativelike conformations. The smallest backbone root mean-square deviation from the crystal structure was 1.1 Å. In the nativelike conformations, the stably formed hydrophobic core was fully dehydrated. Statistical analyses of the simulation trajectories show the following sequential events in folding of HP36: 1), Helix 3 is formed at the earliest stage; 2), the backbone and the side chains near the loop between Helices 2 and 3 take nativelike conformations; and 3), the side-chain packing at the hydrophobic core and the dehydration of the core side chains take place simultaneously at the later stage of folding. This sequence suggests that the initial folding nucleus is not necessarily the same as the hydrophobic core, consistent with a recent experimental ϕ-value analysis.     

Three-dimensional structure of the apo form of the N-terminal EGF-like module of blood coagulation factor X as determined by NMR spectroscopy and simulated folding.

The three-dimensional structure of a 42-residue fragment containing the N-terminal EGF-like module of blood coagulation factor X was determined by means of 2D NMR spectroscopy and computer simulation. The spectroscopic data consisted of 370 NOE distances and 27 dihedral angle constraints. These were used to generate peptide conformations by molecular dynamics simulation. The simulations used a novel functional form for the constraint potentials and were performed with two time steps to ensure rapid execution. Apart from preliminary runs to aid assignment of NOEs, 60 runs resulted in 13 accepted structures, which have two antiparallel beta sheets, no alpha helices, and five tight turns. There is no hydrophobic cluster. The root mean square deviation for the backbone of the 13 conformations is 0.65 +/- 0.11 A against their mean conformation. About half of the side chains have well-defined structure. The overall conformation is similar to that of murine EGF.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

The beta-turn scaffold of tripeptide containing an azaphenylalanine residue

The conformational preferences of azaphenylalanine-containing peptide were investigated using a model compound, Ac-azaPhe-NHMe with ab initio method at the HF/3-21G and HF/6-31G(*) levels, and the seven minimum energy conformations with trans orientation of acetyl group and the 4 minimum energy conformations with cis orientation of acetyl group were found at the HF/6-31G(*) level if their mirror images were not considered. An average backbone dihedral angle of the 11 minimum energy conformations is phi=+/-91 degrees +/-24 degrees , psi =+/-18 degrees +/-10 degrees (or +/-169 degrees +/-8 degrees ), corresponding to the i+2 position of beta-turn (delta(R)) or polyproline II (beta(P)) structure, respectively. The chi(1) angle in the aromatic side chain of azaPhe residue adopts preferentially between +/-60 degrees and +/-130 degrees, which reflect a steric hindrance between the N-terminal carbonyl group or the C-terminal amide group and the aromatic side chain with respect to the configuration of the acetyl group. These conformational preferences of Ac-azaPhe-NHMe predicted theoretically were compared with those of For-Phe-NHMe to characterize the structural role of azaPhe residue. Four tripeptides containing azaPhe residue, Boc-Xaa-azaPhe-Ala-OMe [Xaa=Gly(1), Ala(2), Phe(3), Asn(4)] were designed and synthesized to verify whether the backbone torsion angles of azaPhe reside are still the same as compared with theoretical conformations and how the preceding amino acids of azaPhe residue perturb the beta-turn skeleton in solution. The solution conformations of these tripeptide models containing azaPhe residue were determined in CDCl(3) and DMSO solvents using NMR and molecular modeling techniques. The characteristic NOE patterns, the temperature coefficients of amide protons and small solvent accessibility for the azapeptides 1-4 reveal to adopt the beta-turn structure. The structures of azapeptides containing azaPhe residue from a restrained molecular dynamics simulation indicated that average dihedral angles [(phi(1), psi(1)), (phi(2), psi(2))] of Xaa-azaPhe fragment in azapeptide, Boc-Xaa-azaPhe-Ala-OMe were [(-68 degrees, 135 degrees ), (116 degrees, -1 degrees )], and this implies that the intercalation of an azaPhe residue in tripeptide induces the betaII-turn conformation, and the volume change of a preceding amino acid of azaPhe residue in tripeptides would not perturb seriously the backbone dihedral angle of beta-turn conformation. We believe such information could be critical in designing useful molecules containing azaPhe residue for drug discovery and peptide engineering.     

Conformation of di-n-propylglycine residues (Dpg) in peptides: crystal structures of a type I' beta-turn forming tetrapeptide and an alpha-helical tetradecapeptide.

The crystal structures of two oligopeptides containing di-n-propylglycine (Dpg) residues, Boc-Gly-Dpg-Gly-Leu-OMe (1) and Boc-Val-Ala-Leu-Dpg-Val-Ala-Leu-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe (2) are presented. Peptide 1 adopts a type I'beta-turn conformation with Dpg(2)-Gly(3) at the corner positions. The 14-residue peptide 2 crystallizes with two molecules in the asymmetric unit, both of which adopt alpha-helical conformations stabilized by 11 successive 5 --> 1 hydrogen bonds. In addition, a single 4 --> 1 hydrogen bond is also observed at the N-terminus. All five Dpg residues adopt backbone torsion angles (phi, psi) in the helical region of conformational space. Evaluation of the available structural data on Dpg peptides confirm the correlation between backbone bond angle N-C(alpha)-C' (tau) and the observed backbone phi,psi values. For tau > 106 degrees, helices are observed, while fully extended structures are characterized by tau < 106 degrees. The mean tau values for extended and folded conformations for the Dpg residue are 103.6 degrees +/- 1.7 degrees and 109.9 degrees +/- 2.6 degrees, respectively.     

Molecular dynamics simulation and nmr study of a blood group H trisaccharide

Molecular dynamics simulations in vacuum and solution have been carried out on 2′-α-L -fucosyllactitol, a model for blood group H in conjunction with two-dimensional nmr measurements on the same compound. Three independent starting conformations for the dynamics were chosen from low energy conformations obtained by a ?/ψ grid search. Nine 5 ns vacuum simulations of the trisaccharide were performed, employing three different ways to treat electrostatic interactions for each starting conformation: distance-dependent dielectric with ε = r, constant dielectric with ε = 1, or constant dielectric with ε = 80. In vacuum, transitions of ? and ψ for the α-L -Fuc-(1 → 2)-β-D -Gal element occur in a cooperative manner. The virtual distance obtained for H1 in fucose to H2 in galactose from nuclear Overhauser effect spectroscopy experiments agree with one of the conformations of the trisaccharide in one of the three 100 ps aqueous simulations (?/ψ ca. ?100°/150°), indicating this may be a dominant solution conformation. The rms fluctuations of the ?- and ψ-dihedral angles were ～ 10° for a conformational state, both in the vacuum and the aqueous simulations. For the simulations in vacuum, the agreement with experimental NOE data is reasonable when a constant dielectric of 1 is used (major conformers having ?/ψ ca. ?100°/150° and ?140°/100°), whereas the agreement was poor with a constant dielectric of 80. Translational diffusion coefficients calculated from the simulation of the oligosaccharides were 0.12–0.18 × 10^?5 cm²/s and from nmr measurements 0.27 × 10^?5 cm²/s. © 1994 John Wiley & Sons, Inc.     

Conformational studies of irreversible HIV-1 protease inhibitors containing cis-epoxide as an amide isostere.

We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)2-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isoprop yl)2 where (SMe)Pen(O)2 stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the psi of Phe(cep) is maintained about 130 degrees and the phi angle of (cep)Gly prefers +/- 150 degrees [where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule ('flap water'), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.     

Chromophore distortions in the bacteriorhodopsin photocycle: evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR.

In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.     

Computation of DNA backbone conformations

We present an algorithm for the computation of 2'-deoxyribose-phosphodiester backbone conformations that are stereochemically compatible with a given arrangement of nucleic acid bases in a DNA structure. The algorithm involves the sequential computation of 2'-deoxyribose and phosphodiester conformers (collectively referred to as a backbone 'segment'), beginning at the 5'-end of a DNA strand. Computation of the possible segment conformations is achieved by the initial creation of a fragment library, with each fragment representing a set of bond lengths, bond angles and torsion angles. Following exhaustive searching of sugar conformations, each segment conformation is reduced to a single vector, defined by a specific distance, angle and torsion angle, that allows calculation of the O(1)' position. A given 'allowed' conformation of a backbone segment is determined based on its compatibility with the base positions and with the position of the preceding backbone segment. Initial computation of allowable segment conformations of a strand is followed by the determination of continuous backbone solutions for the strand, beginning at the 3'-end. The algorithm is also able to detect repeating segment conformations that arise in structures containing geometrically repeating dinucleotide steps. To illustrate the utility and properties of the algorithm, we have applied it to a series of experimental DNA structures. Regardless of the conformational complexity of these structures, we are able to compute backbone conformations for each structure. Hence, the algorithm, which is currently implemented within a new computer program NASDAC (Nucleic Acids: Structure, Dynamics and Conformation), should have generally applicability to the computation of DNA structures.     

Structural equilibrium of DNA represented with different force fields.

We have recently indicated preliminary evidence of different equilibrium average structures with the CHARMM and AMBER force fields in explicit solvent molecular dynamics simulations on the DNA duplex d(C5T5) . d(A5G5) (Feig, M. and B.M. Pettitt, 1997, Experiment vs. Force Fields: DNA conformation from molecular dynamics simulations. J. Phys. Chem. B. (101:7361-7363). This paper presents a detailed comparison of DNA structure and dynamics for both force fields from extended simulation times of 10 ns each. Average structures display an A-DNA base geometry with the CHARMM force field and a base geometry that is intermediate between A- and B-DNA with the AMBER force field. The backbone assumes B form on both strands with the AMBER force field, while the CHARMM force field produces heterogeneous structures with the purine strand in A form and the pyrimidine strand in dynamical equilibrium between A and B conformations. The results compare well with experimental data for the cytosine/guanine part but fail to fully reproduce an overall B conformation in the thymine/adenine tract expected from crystallographic data, particularly with the CHARMM force field. Fluctuations between A and B conformations are observed on the nanosecond time scale in both simulations, particularly with the AMBER force field. Different dynamical behavior during the first 4 ns indicates that convergence times of several nanoseconds are necessary to fully establish a dynamical equilibrium in all structural quantities on the time scale of the simulations presented here.     

Conformational analysis of the 3'-5'-cyclic dinucleotide d less than pApA greater than by means of molecular mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.     

Multiple copy sampling in protein loop modeling: computational efficiency and sensitivity to dihedral angle perturbations.

Multiple copy sampling and the bond scaling-relaxation technique are combined to generate 3-dimensional conformations of protein loop segments. The computational efficiency and sensitivity to initial loop copy dispersion are analyzed. The multicopy loop modeling method requires approximately 20-50% of the computational time required by the single-copy method for the various protein segments tested. An analytical formula is proposed to estimate the computational gain prior to carrying out a multicopy simulation. When 7-residue loops within flexible proteins are modeled, each multicopy simulation can sample a set of loop conformations with initial dispersions up to +/- 15 degrees for backbone and +/- 30 degrees for side-chain rotatable dihedral angles. The dispersions are larger for shorter and smaller for longer and/or surface loops. The degree of convergence of loop copies during a simulation can be used to complement commonly used target functions (such as potential energy) for distinguishing between native and misfolded conformations. Furthermore, this convergence also reflects the conformational flexibility of the modeled protein segment. Application to simultaneously building all 6 hypervariable loops of an antibody is discussed.