Conformation and activity of +H2-Pro-Leu-Gly-NH2 and its analogues modified at the leucyl residue

In order to investigate the relation between the conformation and the ability to modulate dopamine receptors, conformational free-energy calculations using an empirical potential (ECEPP/3) and hydration shell model were carried out on the tripeptide ⁺H₂-Pro-Leu-Gly-NH₂ (PLG) and its analogues in which the Leu residue is replaced with Phe, Ahx (L-2-aminohexanoic acid), Ile, Abu and Ala residues in the unhydrated and hydrated states. PLG and two tripeptides possessing Phe and Ahx residues show dopamine receptor modulating activity, while the other tripeptides do not. Irrespective of activities, PLG and its analogues are found to have the high probabilities to form β-bends of types II and I in the unhydrated state. However, in the hydrated state, the β-bend probabilities of the PLG analogues decrease significantly compared with those in the unhydrated state. These results indicate that the β-bend structure is a necessary factor for the PLG analogues to be active, but not a sufficient factor, and that the interactions of water molecules with the backbone may force the tripeptides to be more distorted or extended. The size and the orientation of the hydrophobic moiety of the second residue seem to be of consequence in eliciting the activity of the tripeptides. © Munksgaard 1995.     

Conformational study of Ac-Xaa-Pro-NHMe dipeptides: proline puckering and trans/cis imide bond

The conformational study on 20 Ac-Xaa-Pro-NHMe dipeptides has been carried out using an empirical potential function ECEPP/3 in order to investigate the factors responsible for the preference of proline puckering of the peptides with the trans or cis imide bond preceding the proline. The general conformational preference for down- and up-puckered dipeptides is calculated as trans-down > trans-up > cis-down > cis-up, which is reasonably in accord with that estimated by analyzing X-ray structures of proteins and the result for the single proline residue. The overestimated occurrence of trans-down conformations of proline seems to be caused by excluding long-range interactions that short dipeptides cannot have. The average computed occurrence of dipeptides with cis imide bonds is about 3%, somewhat lower than the value calculated for Ac-Pro-NHMe, which is close to experimental estimates obtained from X-ray structures of proteins. In particular, the interaction of the aromatic side chain of Xaa residue with the proline ring appears not to be strong enough to stabilize the stacked conformations of small dipeptides with cis imide bonds. The propensity to adopt trans or cis imide bond and to form secondary structures of Xaa-Pro sequences is discussed and compared with results obtained from X-ray structures of proteins.     

Characteristic Structural Features of Indolicidin: Effects of the cis‐trans Isomerism on its Conformation

Indolicidin is an antimicrobial peptide showing a broad spectrum of antibacterial and antifungal activities, and according to the cis‐trans isomerism of three Xaa‐Pro peptide bonds, eight different stereoisomers could be distinguished for this peptide. As the cis‐trans isomerism about the Xaa‐Pro peptide bonds was not considered in previous studies, the structural features of distinct stereoisomeric forms were not characterized in detail, so far. In this theoretical study, the influences of cis‐trans isomerism on the conformation of indolicidin were investigated, as well as the typical structural properties of each stereoisomer were determined, focusing on the secondary structures and intramolecular interactions. Based on the results derived from the molecular dynamics simulations, it could be concluded that the eight different stereoisomeric forms of indolicidin adopted characteristic conformational features. Nevertheless, the appearance of various turn structures and intramolecular interactions proved to be dependent on the cis or trans nature of Xaa‐Pro peptide bonds, indicating the relevant role of Pro amino acids in determining the three‐dimensional structure of this peptide.     

Backbone conformations,bend structures,helix structures and other tests of an improved conformational energy program for peptides: ECEPP83

Improvements in the potentials used in the computer program ECEPP (empirical conformational energy program for peptides) and a variety of test cases using a new program (ECEPP83) are described. The changes made in ECEPP are based on recent results of ab initio geometry optimized dipeptide studies, and also on the inability of ECEPP potentials to predict low-energy conformations in regions of Ø-Ø space where experimental β-bends occur. The concept of the thermal ellipsoid is also developed as a method for visualizing correlated conformational motion about energy minima. A series of previously studied bend structures in proteins are examined again using the improved program, ECEPP83. The changes made to the program are found to result in significant modifications in the ordering of computed energy minima, protein bend structures, and in the values of Ø and Ø at the energy minimum for helical homopolymers. Agreement with experiment is improved over that found using ECEPP.     

SPECTROSCOPIC STUDY OF THE CONFORMATIONS OF PROLINE-CONTAINING OLIGOPEPTIDES IN THE CRYSTALLINE STATE AND IN SOLUTION

A conformational study has been carried out on a series of linear proline-containing oligopeptides (ZGP, ZGPL, ZGPLG and ZGPLGP) in both the crystalline state and in DMSO-d₆, solution, using Raman and n.m.r. spectroscopy. The amide I and HI bands in the Raman spectra of the crystalline forms indicate the presence of a type I β-bend conformation in ZGPLG and ZGPLGP, but not in ZGP and ZGPL. This result is in agreement with X-ray data on these mole cules. The Raman spectra of these peptides in solution indicate that more than one conformation is present, i.e. that the β-bend structure of the solid form of ZGPLG and ZGPLGP is destabilized by DMSO-d₆. ¹³C and ¹H n.m.r. data also demonstrate the presence of more than one conformation in ZGP, ZGPL, ZGPLG and ZGPLGP in DMSO-d₆ solution. These isomers differ in their con formation (cis and trans) about their Gly-Pro peptide bonds and possibly about the Cα-C' bond of the C-terminal proline in ZGPLGP. For ZGP, ZGPL, ZGPLG and ZGPLGP, the ensemble of conformations in DMSO-d₆ includes C₅ and C₇ hydrogen-bonded rings; in addition, ZGPLGP may contain a small percentage of a β-bend conformation (at Pro₂-Leuj₃) with trans peptides in both Gly-Pro moieties.     

The β-bend ribbon spiral

The synthesis and conformational analysis in solution (by FTIR absorption and ¹H NMR) and in the crystal state (by X-ray diffraction) of three Hib-containing depsipeptides have been performed. In the crystal state Z-Aib-Hib-Aib-OMe is folded into a type-III β-bend, while the conformation adopted by Z-(Aib-Hib)₂-Aib-OMe is a β-bend ribbon spiral, characterized by two type-III β-bends with Aib(1)-Hib(2) and Aib(3)-Hib(4) as corner residues, respectively. Both independent molecules in the asymmetric unit of t-Boc-L-Ala-Hib-L-Ala-OMe crystals are folded into a type-II β-bend. For the Aib-Hib depsipeptides the conformation adopted in the crystal state is also that largely prevailing in solution, whereas for t-Boc-L-Ala-Hib-L-Ala-OMe the β-bend conformation is significantly less populated in solution. A comparison is also made with: (i) the published crystal-state conformations of fully protected -(Aib)₃^?, -(Aib)₅^?, and -L-Ala-Aib-L-Ala- sequences and the β-bend ribbon spiral generated by (Aib-L-Pro)_n oligomers, and (ii) with the herewith described solution preferred conformation of Z-L-Ala-Aib-L-Ala-OMe. The possible use of Hib as an isosteric replacement for Aib in the design of conformation ally constrained depsipeptides is briefly discussed.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Preferred conformations of a linear RGD tripeptide

Abstract: Conformational study of RGD tripeptides in the nonhydrated and hydrated states was carried out using an empirical potential function ECEPP/3 and the hydration shell model in order to investigate preferred conformations and factors responsible for their stability. RGD tripeptides in the nonhydrated and hydrated states can be interpreted as existing as an ensemble of feasible conformations rather than as a single dominant conformation from the analysis of distributions of backbone conformations, hydrogen bonds and β‐turns. The different distributions of conformations for the neutral and zwitterionic RGD tripeptides in both states may indicate that the conformation of the RGD tripeptide is liable to depend on solvent polarity and pH values. β‐Turn populations for the neutral tripeptide in both states are reasonably consistent with NMR measurements on linear RGD‐containing peptides. The degradation of RGD tripeptide seems to be attributed mainly to the hydrogen bonds between the Asp side‐chain and the backbone of Asp residue or C‐terminal NHMe group, rather than to the flexible backbones of Gly and Asp residues.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.     

Peptidyl‐prolyl isomerases: Functionality and potential therapeutic targets in cardiovascular disease

Peptidyl‐prolyl cis/trans isomerases (PPIases) are a conserved group of enzymes that catalyse the conversion between cis and trans conformations of proline imidic peptide bonds. These enzymes play critical roles in regulatory mechanisms of cellular function and pathophysiology of disease. There are three different classes of PPIases and increasing interest in the development of specific PPIase inhibitors. Cyclosporine A, FK506, rapamycin and juglone are known PPIase inhibitors. Herein, we review recent advances in elucidating the role and regulation of the PPIase family in vascular disease. We focus on peptidyl‐prolyl cis/trans isomerase NIMA‐interacting 1 (Pin1), an important member of the PPIase family that plays a role in cell cycle progression, gene expression, cell signalling and cell proliferation. In addition, Pin1 may be involved in atherosclerosis. The unique role of Pin1 as a molecular switch that impacts on multiple downstream pathways necessitates the evaluation of a highly specific Pin1 inhibitor to aid in potential therapeutic drug discovery.     

Conformation and hydration of aspartame

Conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the neutral, acidic, zwitterionic, and basic forms of aspartame in the hydrated state. The results indicate that as the molecule becomes more charged, the number of low energy conformations becomes smaller and the molecule becomes less flexible. The calculated free energies of hydration of charged aspartames show that hydration has a significant effect on the conformation in solution. Only two feasible conformations were found for the zwitterionic form, and these are consistent with the conformations deduced from NMR and X-ray diffraction experiments. The calculated free energy difference between these two conformations was 1.25 kcal/mol. The less favored of the two solvated conformations can be expected to be stabilized by hydrophobic interaction of the phenyl groups in the crystal.     

CONFORMATIONAL STUDIES ON SEQUENTIAL POLYPEPTIDES:

The conformational properties of (Pro-Phe-Gly)_n and (Phe-Pro-Gly)_n were investigated both in the solid state and in solution. In solid state X-ray diffraction patterns seem to indicate that (Pro-Phe-Gly)_n assumes a single chain triple helical structure. In solution, by circular dichroism studies, it was possible to show the existence of β-bends in the presence of ethylene glycol. Similar studies carried out on (Phe-Pro-Gly)_n showed no collagen or polyproline 11–like structure in the solid state. In solution the β-bend formation was apparent in all the solvent systems studied.     

N-Methyl peptides

The natural occurrence of N-methyl peptides in various plant metabolites has made N-methylation a subtle and attractive possible modification for structure-activity relationship studies of endogeneous peptides. However, little is known about the conformational specificity induced by the N-methylation of a given peptide, and particularly concerning the β-turn conformation. A spectroscopic investigation (i.r., n.m.r., CD) and X-ray diffraction experiments have been carried out on tBuCO-X-Me-Y-NHMe blocked dipeptides (X = Gly, L-Ala, L-Pro, and Y = Gly, and L- or D-Ala, Leu, Phe) with reference to the homologous desmethylated species. The influence of the N-methylation on conformation depends to a large extent on the chirality of the X and Y residues. Homochiral sequences are the most affected, with a strong preference for the βVI-folded conformation containing a middle cis amide bond. Heterochiral sequences are essentially unaffected and retain the βII-folded conformation with a trans middle amide bond. Glycine-containing sequences undergo a more complex perturbation according to the X or Y position of the Gly residue. The available data for larger N-methyl peptides are consistent with our observations, suggesting that these simple dipeptides well reflect the conformational perturbations induced by N-methylation on the β-turn conformation.     

Simulations of conformers of tuftsin and a cyclic tuftsin analog

The conformational properties of the configurational isomers of tuftsin, a linear tetrapeptide with the sequence Thr-Lys-Pro-Arg, were investigated with six 1 ns molecular dynamics simulations in explicit water and in a 1.0 M NaCl solution. The average conformation of the cis isomer is a type VI β-turn. Our results indicate that water-peptide hydrogen bonding, in addition to intramolecular hydrogen bonds, stabilizes the cis conformer. The trans isomer is neither a β- nor a γ-turn. Results are compared with parallel studies on a cyclic analog of tuftsin, cyclo(Thr-Lys-Pro-Arg-Gly). The addition of salt does not influence the backbone conformation of the peptide. Differences between the structures are confined to the side-chain orientations of the Lys and Arg residues. © Munksgaard 1995.     

The receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related δ-opioid antagonists contains all trans peptide bonds

Two different models for the receptor-bound conformation of δ-opioid peptide antagonists containing the N-terminal dipeptide segment H-Tyr-Tic (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic² aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ-antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic² residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H-Tyrψ[CH₂NH]. Tic-Phe-Phe-OH and H-Tyrψ[CH₂NH]. MeTic-Phe-Phe-OH (MeTic = 3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ-opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low-energy conformations in which the torsional angle (“ω₁”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low-energy conformations in which “ω₁” had a value of 0°(cis conformation). Conformers with that same bond in the gauche- conformation (“ω₁”= -60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor-bound conformation of δ-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.     

Correlation of β-bend conformations of tetrapeptides with their activities in CD4-receptor binding assays

Conformational analysis, based on ECEPP (Empirical Conformational Energy Program for Peptides) using the chain build-up procedure, was applied to determine the low-energy conformations for a series of tetrapeptides. The tetrapeptides are components of larger peptides which have been found to bind to the CD4 receptor of monocytes. Several previous studies have implicated the tetrapeptide units investigated here as being critical to the biological activities of the full peptides. Five such tetrapeptides were studied: Ser-Ser-Asn-Tyr (from ribonuclease A), Thr-Thr-Asn-Tyr (from peptide T, known to block human immunodeficiency virus from attaching to CD4⁺ T cells), Thr-Ile-Asn-Tyr (from polio virus coat protein, which is less active than the other peptides in binding to CD4 receptors), Ser-Ser-Ala-Tyr (from the gp 120 coat protein of human immunodeficiency virus, a variant of the peptide T sequence, active in blocking viral attachment to CD4⁺ cells), and the tetrapeptide from an active synthetic pentapeptide, Asn-Thr-Lys-Tyr (from Asn-Thr-Lys-Tyr-Thr). Using a 7 kcal/mol cutoff, the low-energy conformations for each peptide were computed. Approximately 20000 conformations were computed for each tetrapeptide. Residue probability profiles were determined for each tetrapeptide. All tetrapeptides except for the polio sequence showed flexibility in the sense that many low-energy conformations were possible. In previous studies, it was postulated that the critical tetrapeptide units would adopt conformations similar to the one observed in a segment of ribonuclease A, residues 22–25, a β-bend, which is part of an octapeptide segment (residues 19–26) that is homologous to the sequence of peptide T. It was found that the four most active tetrapeptides exhibited 5–10 times the probability of being superimposed on the native structure of the 22–25 segment of ribonuclease A as the less active polio tetrapeptide. The results are compatible with the hypothesis that peptide T adopts the native ribonuclease β-bend (at residues 22–25) for its Thr-Thr-Asn-Tyr sequence.     

Stereochemical studies of cyclic peptides.

Conformational variety in cyclic tripeptides was examined using a combination of stereochemical and energy minimization methods. Four different conformations were found possible: (i) a threefold symmetrical one with all peptide units in cis configuration, with very little nonplanarity; (ii) an asymmetrical one with all cis units and with a larger nonplanarity (ccc); and (iii) two possible conformations with two cis and one trans peptide unit, (cct)₁ and (cct)₂. All these conformations can accommodate both sarcosyl and prolyl residues, which are known to occur in cis peptide units. The symmetric and the (cct)₂ conformations can accommodate only a homo-isomeric sequence of prolyl residues, and the other two asymmetric conformations (ccc) and (cct)₁ can accommodate hetero-isomeric sequence of Pro. In order to relieve some severe steric hindrances cis peptide units show a need-based flexibility in the peptide bond angles. All the four conformations are known to occur in solid state as well and the results obtained compare reasonably well with the available crystal structure information.     

Vitamin K-dependent carboxylase

The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a Boc-protected amino group and a methyl ester group and in position 3 by a free carboxylate group (6–9), has been carried out in an aqueous environment, by ¹H and ¹³C NMR spectroscopy, and molecular dynamics (MD). These compounds have been shown to be weak competitive inhibitors (K_i? 20–65 mM) of the vitamin K-dependent carboxylation of Boc-Glu-OMe in rat liver microsomes independently of their ring size and stereochemical features. However, the cyclic trans isomers have been found more active than the cis ones and Boc-trans-C5-OMe (9) is the most potent inhibitor in the series (cis and trans isomers are defined by the relative arrangement of the carboxyl functions). Such cyclic glutamyl derivatives may provide valuable informations on the preferred bioactive conformations of synthetic glutamyl substrates at the active site of the carboxylase. In aqueous solution, the Boc-cis- and trans-C6 esters exhibit chair conformations with exclusively equatorial and axial substituent positions, while the Boc-cis- and trans-C5 compounds may display envelope E or ‘twist’ T conformations with the substituents in the following positions. equatorial, axial and isoclinal. For each compound, the conformations resulting from NMR and MD data were analyzed and classified according to the dihedral angles %1 and %2, the distances of functional groups, and the spatial charge distribution involving the free carboxyl group. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. These results are discussed in relation with the carboxylase inhibitory activity of the analogues, and a spatial disposition of the glutamyl side chain that could be recognized by the carboxylase is deduced. © Munksgaard 1997.     

CONFORMATIONAL CHARACTERISTICS OF THE N-ACETYL-N‘-METHYLAMIDES OF THE FOUR (LYS,TYR) DIPEPTIDES

The conformational properties of the N-acetyl-N'-methylamides of the dipeptides lysyl-lysine, lysyl-tyrosine, tyrosyl-lysine, and tyrosyl-tyrosine were studied by means of conformational energy calculations, by n.m.r. measurements in deuterated dimethylsulfoxide, and by circular dichroism in water, methanol, dioxane-water, and trifluoroethanol. Since these four dipeptides occur occasionally as bends in proteins, it was of interest to see whether short-range interactions, acting within the terminally blocked dipeptides, are sufficient to stabilize bend conformations significantly over other conformations. It was found that the four dipeptides exist as ensembles of conformations in solution. Therefore, it appears that longer-range interactions, such as those present in proteins, are required if bend conformations of these dipeptide sequences are to exist as stable conformations. Three of the dipeptides behave rather similarly. Both the CD and the n.m.r. experiments and computations indicate that the fourth (Lys-Tyr) differs from the others. It has a preference for compact conformations that appear to be stabilized by strong favorable interactions, primarily hydrogen bonds, between the tyrosyl and the lysyl side chains. The computations suggest that the presence of these interactions, and hence the existence of preferred conformations, is strongly solvent-dependent, and that these interactions are weakened in aqueous solution.     

Empirical rules predicting conformation of cyclic tetrapeptides from primary structure

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: 1. A conformation allowing an intramolecular hydrogen bond (IMHB) of γ-turn is preferred, and an ester bond always adopts a trans form. 2. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. 3. The carbonyl group acylating a d proline or an N-methyl-d -amino acid residue is oriented to the lower side of the ring, forming a cis bond. 4. The lddl configurational sequence adopts a cis-trans-cis-trans backbone with C_i symmetry. 5. A glycine residue behaves as a d residue in an l -peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo (-l -Pro-l -Leu-d -Tyr(Me)-l -Ile-)(1) and cyclo (-l -Pro-d -Leu-d -Tyr(Me)-l -Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a γ-turn IMHB, and 2 has a cis-trans-cis-trans backbone with C_i symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (ld ) and cis (dd ) secondary amide bonds.