Evaluation of the conformational propensities of peptide isosteres as a basis for selecting bioactive pseudopeptides.

Our aim was to compare the repertoires of conformers formed by the model zwitterionic peptides AA and AAA in aqueous solution with the conformational profiles of a range of their peptide isosteres, so as to facilitate selection of isosteres for synthesis and testing as biologically stable surrogates of bioactive di- and tripeptides. Comparisons were based upon the results of conformational analysis using a random search approach implemented within the SYBYL molecular modelling package, using zwitterionic molecules, simulated aqueous solvation using a dielectric constant of 80 and allowing all torsions to vary. For each compound, individual conformers were grouped on the basis of specific combinations of psi, phi and omega torsions and, using their energies, the aggregated percentage for each group was calculated using a Boltzmann distribution and displayed using a 3D pseudo Ramachandran plot relating percentage conformer to psi and phi torsions. Retroamide, N-methylamide and thioamide isosteres showed the best match to natural peptides and to the molecular recognition parameters defined for substrates of peptide transporters. The results should aid rational design of therapeutic agents in various areas, e.g. oral delivery of drugs by peptide transporters and of peptidase inhibitors. This approach may usefully be applied to various biochemical and pharmaceutical topics.     

Theoretical studies on the conformation of saccharides. XIV. Structure and conformational properties of the glycosylamines

The 2-methylaminotetrahydropyran was used as a model to study conformational properties of the N-glycosidic linkage in glycosylamines. Relaxed two-dimensional conformational (phi, psi) maps in 20 solvents were calculated by a method in which the total energy is divided into the energy of the isolated molecule and the solvation energy. Molecular geometry optimization has been carried out for each conformer using the quantum chemical method PCILO. The calculated variations of the geometry are consistent with the results obtained by the statistical analysis of available experimental data retrieved from the Cambridge Structural Database. The calculated abundances of conformers show that the polarity of the solvent has little effect on the anomeric ratio, and the form having the methylamino group equatorial is favored in all considered solvents.     

Predominant torsional forms adopted by dipeptide conformers in solution: parameters for molecular recognition.

The present paper describes the predominant conformational forms adopted by dipeptides in aqueous solution. More than 50 dipeptides were subjected to conformational analysis using SYBYL Random Search. The resultant collections of conformers for individual dipeptides, for small groups with related side chain residues and for large groups of about 50 dipeptides were visualized graphically and analysed using a novel three-dimensional pseudo-Ramachandran plot. The distribution of conformers, weighted according to the percentage of each in the total conformer pool, was found to be restricted to nine main combinations of backbone psi (psi) and phi (phi) torsion angles. The preferred psi values were in sectors A7 (+150 degrees to +/-180 degrees), A10 (+60 degrees to +90 degrees) and A4 (-60 degrees to -90 degrees), and these were combined with preferred phi values in sectors B12 (-150 degrees to +/-180 degrees), B9 (-60 degrees to -90 degrees) and B2 (+30 degrees to +60 degrees). These combinations of psi and phi values are distinct from those found in common secondary structures of proteins. These results show that although dipeptides can each adopt many conformations in solution, each possesses a profile of common conformers that is quantifiable. A similarly weighted distribution of dipeptide conformers according to distance between amino-terminal nitrogen and carboxyl-terminal carbon shows how the preferred combinations of backbone torsional angles result in particular N-C geometries for the conformers. This approach gives insight into the important conformational parameters of dipeptides that provide the basis for their molecular recognition as substrates by widely distributed peptide transporters. It offers a basis for the rational design of peptide-based bioactive compounds able to exploit these transporters for targeting and delivery.     

Disaccharide conformational maps: 3D contours or 2D plots?

The potential energy surfaces of several alpha-(1-->3)- and beta-(1-->4)-linked disaccharides were obtained and plotted in terms of energy versus psi glycosidic angle. These plots were compared to those obtained previously in the way of the usual 3D contour maps, which relate the energy with the two glycosidic angles (phi and psi). Given the usually small variations of the phi angle in the low-energy regions (at least using MM3), both kinds of graphs lead to similar conclusions concerning flexibility measurements by two different methods and assessment of the effects of sulfation and/or hydroxyl group orientation. Only second-order effects were found with some sulfated disaccharides, not changing the general conclusions. The computational efforts required to produce those plots are smaller, and the plots are easier to interpret. Besides, the conversion of a 3D map into a 2D plot leaves the possibility of constructing 3D maps of carbohydrates including a second variable different to phi, e.g., the second psi angle of a trisaccharide or the omega angle of a 6-linked disaccharide.     

Conformational investigation of alpha, beta-dehydropeptides. XI. Molecular and crystal structure of Ac-(Z)-deltaPhe-NMe2 as compared to those of related molecules.

A series of three homologous dimethyldiamides Ac-(Z)-deltaPhe-NMe2, Ac-L-Phe-NMe2 and Ac-DL-Phe-NMe2 have been synthesized and their structures determined from single-crystal X-ray diffraction data. To learn more about the conformational preferences of the compounds studied, the fully relaxed phi, psi conformational energy maps on the free molecules of Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 were obtained with the HF/3-21G method and the calculated minima re-optimized with the DFT/B3LYP/6-31G** method. The crystal state results have been compared with the literature data. The studied dimethyldiamide Ac-deltaXaa-NMe2 combines the double bond in positions alpha, beta and the C-terminal tertiary amide within one molecule. As the representative probe with deltaXaa = deltaAla, (Z)-deltaLeu and (Z)-deltaPhe shows, in the solid state they adopt the conservative conformation with phi, psi approximately -45 degrees, approximately 130 degrees and with a non-planar tertiary amide bond, whatever the packing forces are. This conformation is located on the Ramachandran map in region H/F, which is of high-energy for common amino acids, but not so readily accessible to them. The free molecule calculations on Ac-deltaAla-NMe2 and Ac-(Z)-deltaPhe-NMe2 reveal that, in spite of dissimilar overall conformational profiles of these molecules, this structure is one of their low-energy conformers and for Ac-(Z)-deltaPhe-NMe2 it constitutes the global minimum. So, the theoretical results corroborate those experimental results proving that this structure is robust enough to avoid conformational distortion due to packing forces. In contrast to Ac-deltaXaa-NMe2, the saturated Ac-L/DL-Xaa-NMe2 shows the constancy of the associative patterns but do not prefer any molecular structure in the solid state.     

Empirical torsional potential functions from protein structure data. Phi- and psi-potentials for non-glycyl amino acid residues.

The torsional potential functions Vt(phi) and Vt(psi) around single bonds N--C alpha and C alpha--C, which can be used in conformational studies of oligopeptides, polypeptides and proteins, have been derived, using crystal structure data of 22 globular proteins, fitting the observed distribution in the (phi, psi)-plane with the value of Vtot(phi, psi), using the Boltzmann distribution. The averaged torsional potential functions, obtained from various amino acid residues in L-configuration, are Vt(phi) = 1.0 cos (phi + 60 degrees); Vt(psi) = 0.5 cos (psi + 60 degrees) - 1.0 cos (2 psi + 30 degrees) - 0.5 cos (3 psi + 30 degrees). The dipeptide energy maps Vtot(phi, psi) obtained using these functions, instead of the normally accepted torsional functions, were found to explain various observations, such as the absence of the left-handed alpha helix and the C7 conformation, and the relatively high density of points near the line psi = 0 degrees. These functions derived from observational data on protein structures, will, it is hoped, explain various previously unexplained facts in polypeptide conformation.     

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.     

Predominant torsional forms adopted by oligopeptide conformers in solution: parameters for molecular recognition.

In this paper, we describe the predominant conformational forms adopted by tripeptides and higher oligopeptides in aqueous solution. About 50 tripeptides and almost 20 higher oligopeptides (4-6 residues) were subjected to conformational analysis using SYBYL Random Search. As with dipeptides (Grail BM, Payne JW. J. Peptide Sci. 2000; 6: 186-199), both tripeptides and higher oligopeptides were found to occupy relatively few combinations of psi-phi space that were distinct from those associated with predominant protein secondary structures (e.g. helices and beta-sheets). Again, the preferred psi (psi) values for the first residue (i - 1) were in sectors encompassed by the ranges from +150 degrees to +/-180 degrees, +60 degrees to +90 degrees and -60 degrees to -90 degrees, which were combined with preferred phi (phi) values for the second residue (i) in sectors with ranges from -150 degrees to +/-180 degrees, -60 degrees to -90 degrees and +30 degrees to +60 degrees. It was notable that tripeptides and, to a greater extent, higher oligopeptides adopted an initial psi (psi) (Tor2) from +150 degrees to +/-180 degrees. For tripeptides, their N-C distances (distance between N-terminal nitrogen and C-terminal carbon atoms) distribute about 6.5 A to give shorter, 'folded' conformers that are similar in length to dipeptides, and longer, 'extended' conformers that are distinct. Furthermore, for higher oligopeptides, their N-C distances did not increment in relation to their increasing number of residues and short, 'folded' conformers were still present. These findings have a bearing upon the recognition of these molecules as substrates for widely distributed peptidases and peptide transporters.     

Conformation of the O-specific polysaccharide of Shigella dysenteriae type 1: molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp.

The O-specific polysaccharide of Shigella dysenteriae type 1, which has the repeating tetrasaccharide unit -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcNAcp-(1--> (A-B-C-D), is a major virulence factor, and it is believed that antibodies against this polysaccharide confer protection to the host. The conformational properties of fragments of this O-antigen were explored using systematic search with a modified HSEA method (GLYCAN) and with molecular mechanics MM3(96). The results show that the alpha-D-Gal-(1-->3)-alpha-D-GlcNAc linkage adopts two favored conformations, phi/psi approximately equal to -40 degrees /-30 degrees (I) and approximately 15 degrees /30 degrees (II), whereas the other glycosidic linkages only have a single favored phi/psi conformational range. MM3 indicates that the trisaccharide B-C-D and tetrasaccharides containing the B-C-D moiety exist as two different conformers, distinguished by the conformations I and II of the C-D linkage. For the pentasaccharide A-B-C-D-A' and longer fragments, the calculations show preference for the C-D conformation II. These results can explain previously reported nuclear magnetic resonance data. The pentasaccharide in its favored conformation II is sharply bent, with the galactose residue exposed at the vertex. This hairpin conformation of the pentasaccharide was successfully docked with the binding site of a monoclonal IgM antibody (E3707 E9) that had been homology modeled from known crystal structures. For fragments made of repetitive tetrasaccharide units, the hairpin conformation leads to a left-handed helical structure with the galactose residues protruding radially at the helix surface. This arrangement results in a pronounced exposure of the galactose and also the adjacent rhamnose in each repeating unit, which is consistent with the known role of the as alpha-L-Rhap-(1-->2)-alpha-D-Galp moiety as a major antigenic epitope of this O-specific polysaccharide.     

Comparison of a QM/MM force field and molecular mechanics force fields in simulations of alanine and glycine "dipeptides" (Ace-Ala-Nme and Ace-Gly-Nme) in water in relation to the problem of modeling the unfolded peptide backbone in solution

We compare the conformational distributions of Ace-Ala-Nme and Ace-Gly-Nme sampled in long simulations with several molecular mechanics (MM) force fields and with a fast combined quantum mechanics/molecular mechanics (QM/MM) force field, in which the solute's intramolecular energy and forces are calculated with the self-consistent charge density functional tight binding method (SCCDFTB), and the solvent is represented by either one of the well-known SPC and TIP3P models. All MM force fields give two main states for Ace-Ala-Nme, beta and alpha separated by free energy barriers, but the ratio in which these are sampled varies by a factor of 30, from a high in favor of beta of 6 to a low of 1/5. The frequency of transitions between states is particularly low with the amber and charmm force fields, for which the distributions are noticeably narrower, and the energy barriers between states higher. The lower of the two barriers lies between alpha and beta at values of psi near 0 for all MM simulations except for charmm22. The results of the QM/MM simulations vary less with the choice of MM force field; the ratio beta/alpha varies between 1.5 and 2.2, the easy pass lies at psi near 0, and transitions between states are more frequent than for amber and charmm, but less frequent than for cedar. For Ace-Gly-Nme, all force fields locate a diffuse stable region around phi = pi and psi = pi, whereas the amber force field gives two additional densely sampled states near phi = +/-100 degrees and psi = 0, which are also found with the QM/MM force field. For both solutes, the distribution from the QM/MM simulation shows greater similarity with the distribution in high-resolution protein structures than is the case for any of the MM simulations.     

Theoretical conformational analysis of methylamide of N-acetyl-L-lysine]

The spatial structure of the methylamide of N-acetyl-L-lysine has been analysed taking into account non-bonded and electrostatic interactions, torsional energy, bond angles distortion and hydrogen bonding. Conformational capacities of the backbone and mutual dependence of spatial structures of the backbone and the side chain was described by conformational maps obtained by energy minimisation, the dihedral angles and the bond angles of the side chain being varied for every phi, psi point. Every possible combination for phi, psi, x1-x5-angles was used corresponding to the stable form of the backbone and to torsion potential minima of the initial approximations in the calculation of preferred conformations of the molecule. Comparisons are made between stable forms of the methylamide of N-acetyl-L-lysine and Lys residues in proteins with known structure.     

Conformational analysis of inulobiose by molecular mechanics

Conformational energies for inulobiose [beta-D-fructofuranosyl-(2----1)-beta-D-fructofuranoside], a model for inulin, were computed with the molecular mechanics program MMP2(85). The torsion angles of the three linkage bonds were driven in 20 degree increments, and the steric energy of all other parameters was minimized. The linkage torsion angles defined by C-1'-C-2'-O-C-1 (phi) and O-C-1-C-2-O-2 (omega) have minima at +60 degrees and -60 degrees, respectively, regardless of side group orientation; accessible minima exist at other staggered conformations. The torsion angle at the central bond C-2'-O-1-C-1-C-2 (psi) was approximately 180 degrees in all the low-energy conformers. This appears to be generally true for rings linked by three bonds. The fructofuranose rings initially had low-energy 4/3T conformations (angle of pseudorotation, phi 2 = 265 degrees) that were retained except when the linkage conformations created severe inter-residue conflicts. In those cases, almost all puckerings of the furanose rings were found.     

Conformational subspace in simulation of early-stage protein folding

A probability calculus was used to simulate the early stages of protein folding in ab initio structure prediction. The probabilities of particular phi and psi angles for each of 20 amino acids as they occur in crystal forms of proteins were used to calculate the amount of information necessary for the occurrence of given phi and psi angles to be predicted. It was found that the amount of information needed to predict phi and psi angles with 5 degrees precision is much higher than the amount of information actually carried by individual amino acids in the polypeptide chain. To handle this problem, a limited conformational space for the preliminary search for optimal polypeptide structure is proposed based on a simplified geometrical model of the polypeptide chain and on the probability calculus. These two models, geometric and probabilistic, based on different sources, yield a common conclusion concerning how a limited conformational space can represent an early stage of polypeptide chain-folding simulation. The ribonuclease molecule was used to test the limited conformational space as a tool for modeling early-stage folding.     

Peptide-sandwiched protoporphyrin compounds mimicking hemoprotein structures in solution

A series of covalently bound peptide-protoporphyrin-peptide compounds, also carrying naphthalene (N) to allow a photophysical investigation, were synthesized. Their general formula is P(nN)(2), where P refers to protoporphyrin IX, and n to the number of amino acids in the sequence Boc-Leu-Leu-Lys-(Ala)(x) -Leu-Leu-Lys-OtBu of each backbone chain (x = 0-3; n = x + 6). Their structural features in methanol solution were investigated by ir and CD spectra, and by steady-state and time resolved fluorescence experiments as well. The ir spectra indicate that intramolecularly H-bonded conformations form, and CD data in both methanol and water-methanol mixture suggest the presence of alpha-helix structure. Quenching of excited naphthalene takes place by electronic energy transfer from singlet N* to P ground state. Fluorescence decays coupled with molecular mechanics calculations indicate that two conformers for each dimeric peptide are the major contributors to the observed phenomena. These conformers are characterized by a globular, protein-like structure, where the protoporphyrin resides in a central pocket, while the two N groups are externally situated. Of the four N linkages in the two conformers, three of them attain a very similar steric arrangement around the central P molecule, in terms of both center-to-center distance and mutual orientation, while the fourth experiences a different steric disposition as compared to the others. Experimental photophysical parameters satisfactorily compare with those obtained by theoretical calculations, within the F?rster mechanism for long-range energy transfer, only when the mutual orientation of the chromophores was also taken into account. This implies that interconversion among conformational substates of probes linkages is slow on the time scale of the energy transfer process.     

Anomalously high aggregation level of the polyene antibiotic amphotericin B in acidic medium: implications for the biological action

Amphotericin B (AmB) is a polyene antibiotic used to treat deep-seated mycoses. Both the therapeutic action and the toxic side effects of this drug are dependent on its molecular organization. AmB appears as a zwitterion at neutral pH owing to NH(3)(+) and COO(-) groups. The results obtained with electronic absorption, fluorescence, resonance light scattering and infrared absorption spectroscopic analyses show that in the aqueous medium at pH above 10 AmB appears in the monomeric form owing to the negative net electric charge of the molecule. On the contrary, anomalously high aggregation level has been observed at pH below 2, despite the positive net electric charge. The effect is interpreted in terms of the permanent polarization of the polyene chain at low pH, associated with relative rotational freedom of the charged mycosamine fragment of the molecule. The pH-dependent aggregation of AmB is discussed in aspect of pharmacological action of the drug.     

Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly].

A biologically active analog of beta-casomorphin, H-Tyr-cyclo[D-OrnPheProGly], was studied by theoretical conformational analysis. Random sampling was used to search the conformational space of allowed dihedral angles. Among 53 low-energy conformers with different folding of the peptide cyclic moiety, only those were selected which correspond to the low-energy area of the model linear tripeptide Ac-D-AlaAlaPro-NHMe. This peptide was used as the main chain "template" for the D-OrnPheProGly fragment of the studied cyclopeptide molecule. Only 15 conformers were chosen as potentially biologically active structures. The conformational possibilities of the Phe residue were constrained to the combined (A + G) region of the Ala residue phi,psi-map for linear peptides.     

Dihedral angles of tripeptides in solution directly determined by polarized Raman and FTIR spectroscopy

The amide I mode of the peptide linkage is highly delocalized in peptides and protein segments due to through-bond and through-space vibrationally coupling between adjacent peptide groups. J. Phys. Chem. B. 104:11316-11320) used coherent femtosecond infrared (IR) spectroscopy to determine the excitonic coupling energy and the orientational angle between the transition dipole moments of the interacting amide I modes of cationic tri-alanine in D(2)O. Recently, the same parameters were determined for all protonation states of tri-alanine by analyzing the amide I bands in the respective IR and isotropic Raman spectra (. J. Am. Chem. Soc. 119:1720-1726.). In both studies, the dihedral angles phi and psi were then obtained by utilizing the orientational dependence of the coupling energy obtained from ab initio calculations on tri-glycine in vacuo (. J. Raman Spectrosc. 29:81-86) to obtain an extended 3(1) helix-like structure for the tripeptide. In the present paper, a novel algorithm for the analysis of excitonic coupling between amide I modes is presented, which is based on the approach by Schweitzer-Stenner et al. but avoids the problematic use of results from ab initio calculations. Instead, the dihedral angles are directly determined from infrared and visible polarized Raman spectra. First, the interaction energy and the corresponding degree of wave-function mixing were obtained from the amide I profile in the isotropic Raman spectrum. Second, the depolarization ratios and the amide I profiles in the anisotropic Raman and IR-absorption spectra were used to determine the orientational angle between the peptide planes and the transition dipole moments, respectively. Finally, these two geometric parameters were utilized to determine the dihedral angles phi and psi between the interacting peptide groups. Stable extended conformations with dihedral angles in the beta-sheet region were obtained for all protonation states of tri-alanine, namely phi(+) = -126 degrees, psi(+) = 178 degrees; phi(+/-) = -110 degrees, psi(+/-) = 155 degrees; and phi(-) = -127 degrees, psi(-) = 165 degrees for the cationic, zwitterionic, and anionic state, respectively. These values reflect an extended beta-helix structure. Tri-glycine was found to be much more heterogeneous in that different extended conformers coexist in the cationic and zwitterionic state, which yield a noncoincidence between isotropic and anisotropic Raman scattering. Our study introduces vibrational spectroscopy as a suitable tool for the structure analysis of peptides in solution and tripeptides as suitable model systems for investigating the role of local interactions in determining the propensity of peptide segments for distinct secondary structure motifs.     

Conformational limitations of glycylsarcosine as a prototypic substrate for peptide transporters

Peptide transporters are present in all species to absorb the small peptides that occur ubiquitously as products of proteolysis. The broad substrate specificities of these systems allow them to be exploited therapeutically for delivery of peptidomimetic drugs in microbes and man. To this end, glycylsarcosine is currently used as a standard substrate for assaying peptidomimetic transport by peptide transporters. However, in this study we find it is unsuitable as a general substrate, based on assays of its transport by model bacterial peptide transporters and computer-based conformational analysis of its structure. Of the two generic transporters for di- and tripeptides, exemplified by Dpp and Tpp in Escherichia coli, only Dpp can transport glycylsarcosine. The explanation for this finding came from molecular modelling, which indicated that glycylsarcosine can adopt only a restricted range of conformers compared with typical dipeptides, and that of the conformers with a trans peptide bond, the majority have the specific psi and phi backbone torsion angles needed for molecular recognition and transport by Dpp but none possessed psi and phi torsions required for recognition by Tpp; moreover, 38% of its conformers have cis peptide bonds that are not substrates for any peptide transporter. Thus, using glycylsarcosine as substrate in competition assays with compounds that typically form conformers recognised by both types of peptide transporter will underestimate their transport. These findings have implications for assays of oral availability of peptidomimetic drugs such as beta-lactams, ACE inhibitors and anti-viral compounds, for which glycylsarcosine is routinely used.     

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.