Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.     

Design, synthesis and conformational analysis of gamma-turn peptide mimetics of bradykinin.

Gamma-turns are regular secondary structure elements, found with some frequency in small peptides, that have been implicated in the biologically active conformations of several systems. This report describes the design, synthesis and conformational analysis of a non-peptide gamma-turn mimetic. Low energy conformations of the mimetic system exhibit good conformational agreement with an experimentally observed peptide gamma-turn. The mimetics were incorporated into the nonapeptide bradykinin, for which a gamma-turn, formed by residues Ser 6 to Phe 8, has been hypothesized to be a bioactive conformation. The results indicate that a bioactive conformation of bradykinin may include a reverse turn at this position.     

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL).The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL.The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds.The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.     

The twists and turns of beta-peptides.

Recently, it has been discovered that peptides composed of beta-amino acids are capable of adopting novel secondary structures demonstrating that peptides composed of alpha-amino acids are not unique in their ability to fold into well-defined structures. Cyclic as well as acyclic peptides composed of beta-amino acid residues adopt turn, helical, and sheet-like conformations. Here, we discuss the synthesis and conformational preferences of individual, substituted beta-amino acids as well as the structures that peptides composed of these residues, beta-peptides, may adopt.     

NMR study of cyclic peptides with renin inhibitor activity

Several cyclic analogues of renin inhibitors, based on Glu-D-Phe-Lys motif have been investigated by NMR spectroscopy and molecular dynamics calculations (MD). The 15 membered macrocycle, resulting from Glu and Lys side-chain cyclization, exhibits conformational preference. The structural evidence from NMR shows the presence of hydrogen bond between Lys NH and Glu side-chain carbonyl, resulting in a 10 membered pseudo beta-turn-like structure. The structure of the cyclic moiety is similar in all the peptides, which takes at least two conformations around Calpha-Cbeta in Glu side chain. The restrained MD calculations further support such observations and show that the macrocycle is fairly rigid, with two conformations about the Glu Calpha-Cbeta bond. The linear peptide appendages, which are essential for activity in cyclic peptides, show an extended structure in the beta-region of Ramchandran plot. These calculations also demonstrate that for the most active peptide, two major conformers each exist about the Calpha-CO bond of the Lys, D-Trp and Leu residues. In this peptide, the cyclic moiety presents a negatively charged surface formed due to the carbonyl oxygens, which are thus available to form hydrogen bonds with the receptor. The linear fragment presents further binding sites with a surface which has the hydrophobic side chains of D-Trp, Leu and D-Met on one side and carbonyls on the other side.     

Intramolecularly hydrogen-bonded peptide conformations

Over the past few years the possible occurrence of intramolecularly hydrogen-bonded structures in linear and cyclic peptides has attracted increasing attention. In this review emphasis is given to solid-state studies, particularly by X-ray diffraction and infrared absorption techniques. Conformational energy calculations are also considered. The discussion is focused both on model peptides and biological activity polypeptide molecules. The tetrapeptide system (Formula: see text), examined allows one to discuss the extended C5 structure and the various folded conformations, namely the C7 (gamma-turn), C8, C10 (beta-turn), C11, and C13 conformations. The four latter forms may include cis peptide configurations. The oxy-analogs to the C7, C10, and C13 conformations and structures containing bifurcated hydrogen bonds are also discussed. The last sections describe intramolecularly hydrogen-bonded peptide structures involving: (1) a side-chain group, (2) the N-protecting group (in synthetic model compounds), and (3) a beta-amino acid.     

Comparison of three gamma-turn mimetic scaffolds incorporated into angiotensin II

Rigidification of peptides by cyclization and iterative incorporation of well-defined secondary structure mimetics constitutes one approach to the design of non-peptidergic structures with better defined conformations. We herein present the synthesis of a potential gamma-turn mimetic scaffold, and its incorporation in the 3-5 position of angiotensin II. Two analogues of angiotensin II (Ang II) incorporating this 1,3,5-trisubstituted benzene gamma-turn scaffold were synthesized. Evaluation of the compounds in a radioligand binding assay showed that they lacked affinity to the AT1 receptor. To rationalize these results a geometrical and electrostatical comparison with Ang II analogues encompassing a bicyclic scaffold that delivered inactive pseudo peptides and an azepine scaffold producing highly active ligands was made. This analysis did not provide a clear rationale for the inactivity of the benzene gamma-turn scaffolds.     

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.     

Conformational analysis of enkephalin analogs containing a disulfide bond. Models for delta- and mu-receptor opioid agonists

Conformational analysis of the cyclic opioids H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and H-Tyr-D-Cys-Gly-Phe-D-Cys-OH (DCDCE) have been performed using the AMBER program. DPDPE is considerably more selective for delta-receptors than DCDCE. Using the RNGCFM program, a large number of ways were found to close the 14-membered disulfide-containing ring structure. However, intramolecular hydrogen bonds were only possible in gamma-turn and inverse gamma-turn conformations centered on the glycine residue which were associated with opposite chiralities of the disulfide bond. With the cyclic part of the molecules in either a gamma-turn or inverse gamma-turn, a systematic conformational analysis was performed on the tyrosine and phenylalanine sidechains. This showed that conformers with the tyrosine and phenylalanine phenyl rings in the vicinity of the disulfide bond were preferred due to attractive van der Waals forces. For DPDPE, however, this was only possible with a positive dihedral angle for the disulfide bond due to the presence of the beta-carbon methyls of Pen2. In contrast, these preferred conformers were possible with both chiralities of the disulfide bond in DCDCE. Conformational entropies and free energies were computed from the translational, rotational, and vibrational energy levels available to each conformer. The conformational entropies were found to vary significantly and to result in a re-ordering of the lowest energy minima. Based on these conformational differences in DPDPE and DCDCE and their differing pharmacological selectivities, tentative conformational preferences for delta- and mu-receptor opioid peptides are proposed.     

Beta-alanine containing peptides: a novel molecular tool for the design of gamma-turns.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution conformational characterization of the cyclic tetrapeptide cyclo-(L-Pro-beta-Ala-L-Pro-beta-Ala). This peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide (DCCI). The compound crystallizes in the orthorombic space group P2(1)2(1)2(1) from ethyl acetate. All peptide bonds are trans. The molecular conformation is stabilized by two intramolecular hydrogen bonds between the CO and NH groups of the two beta-alanine residues. These hydrogen bonds take place in a C7 structure in which both proline residues occupy the 2 position of an inverse gamma-turn. The two beta-alanine residues have a typical folded conformation (around the C alpha-C beta bond) observed in other cyclic peptides containing this residue. A detailed 1H-nmr analysis in CD3CN solution has been carried out. The molecule assumes a twofold symmetry in solution with a molecular conformation consistent with that observed in the solid state.     

Reversible scaling of dihedral angle barriers during molecular dynamics to improve structure prediction of cyclic peptides.

Peptide cyclization or chemical cross-linking has frequently been used to restrict the conformational freedom of a peptide, for example, to enhance its capacity for selective binding to a target receptor molecule. Structure prediction of cyclic peptides is important to evaluate possible conformations prior to synthesis. Because of the conformational constraints imposed by cyclization low energy conformations of cyclic peptides can be separated by large energy barriers. In order to improve the conformational search properties of molecular dynamics (MD) simulations a potential scaling method has been designed. The approach consists of several consecutive MD simulations with a specific lowering of dihedral energy barriers and reduced nonbonded interactions between atoms separated by three atoms followed by gradually scaling the potential until the original barriers are reached. Application to four cyclic penta- and hexa-peptide test cases and a protein loop of known structure indicates that the potential scaling method is more efficient and faster in locating low energy conformations than standard MD simulations. Combined with a generalized Born implicit solvation model the low energy cyclic peptide conformations and the loop structure are in good agreement with experiment. Applications in the presence of explicit water molecules during the simulations showed also improved convergence to structures close to experiment compared with regular MD.     

Conformations of beta-amino acid residues in peptides: X-ray diffraction studies of peptides containing the achiral residue 1-aminocyclohexaneacetic acid, beta3,3Ac6c

The conformational preferences of the 3,3-disubstituted beta-amino acid residue, 1-aminocyclohexaneacetic acid (beta3,3Ac6c) have been investigated by determining the crystal structures of the parent amino acid, the hydrochloride derivative, 10 protected derivatives and di and tripeptides. The symmetrical cyclohexyl substituent at the beta-position restricts the values of the torsion angles phi (N--C(beta)) and theta (C(beta)--C(alpha)) to approximately gauche values (+/-60 degrees ). Relatively few intramolecularly hydrogen bonded conformations are observed. In the dipeptide Boc-beta(3,3)Ac6c-beta(3,3)Ac6c-NHMe a C6 hydrogen bond is observed. In Piv-Pro-beta(3,3)Ac6c-NHMe a C11 hydrogen bonded hybrid alphabeta turn is characterized. In a majority of cases the amino group occupies the axial position in the cyclohexane ring. The conformations observed are compared with crystallographically observed structures for other beta-residues, including beta(2,2)Ac6c.     

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.     

Molecular mechanics investigation of the flexibility of some cyclic peptides

Conformation space near the crystal conformations of proline-containing cyclic octapeptides and cyclic hexapeptides of C2 sequence symmetry, e.g. cyclo-(Gly-Pro-D-Phe)2 and cyclo-(D-Ala-Gly-Pro-D-Phe)2, was explored using molecular mechanics. Conformations found in crystals were energy minimized, distortions were introduced by systematically fixing backbone dihedral angles at individual residues, and nearby energy-minimized conformations were then located. Interatomic distances and dihedral angles were examined in the conformations within a few kilocalories of the most stable conformation. A common form of flexibility was found to involve libration of amide planes. Among the peptides examined, the cyclic hexapeptides were found to have greater freedom than the cyclic octapeptides, and cyclo-(D-Ala-Gly-Pro-D-Phe)2 was found to be more rigid than cyclo-(D-Ala-Gly-Pro-Phe)2.     

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.     

RNA conformational classes

RNA exhibits a large diversity of conformations. Three thousand nucleotides of 23S and 5S ribosomal RNA from a structure of the large ribosomal subunit were analyzed in order to classify their conformations. Fourier averaging of the six 3D distributions of torsion angles and analyses of the resulting pseudo electron maps, followed by clustering of the preferred combinations of torsion angles were performed on this dataset. Eighteen non-A-type conformations and 14 A-RNA related conformations were discovered and their torsion angles were determined; their Cartesian coordinates are available.     

Vibrational analysis of peptides, polypeptides, and proteins. XXX. Normal mode analyses of gamma-turns

The normal modes have been calculated for three kinds of low energy gamma-turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a gamma-turn, a mirror-related gamma-turn, and an inverse gamma-turn of CH3-CO-(L-Ala)n-NH-CH3, with n = 3 and n = 5, and for certain 14C and 15N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and gamma-turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.     

Conformations of cyclic 3',5'-nucleotides. Effect of the base on the synanti conformer distribution

The furanose and the phosphate rings of cyclic 3′,5′-nucleotides are locked in the ₄T³ and chair conformations respectively. The only variable which shows major conformational flexibility in these molecules is the rotation about the glycosyl bond which describes the orientation of the base relative to the sugar-phosphate bicyclic system. The glycosyl torsion angle has been analyzed for cyclic nucleotides with different purine and pyrimidine bases by use of conformational energy calculations. The results indicate that all the pyrimidine bases, U, T and C show a very strong energetic preference for the anti range of conformations. The calculations predict that among cyclic 3′,5′-purine nucleotides cyclic GMP and cyclic IMP favor the syn conformation to the anti by 95:5 and 70:30 respectively, while cyclic AMP shows a preference for the anti conformation to syn by 70:30. Thus the purines show a greater probability for the syn conformation than the pyrimidines in cyclic 3′,5′-nucleotides.     

Conformational analysis of [Cpp1, Sar7, Arg8] vasopressin by 1H-NMR spectroscopy and molecular mechanics calculations.

A combined 1H-NMR and molecular mechanics study of [Cpp1, Sar7]AVP was performed in order to select the most probable conformations in DMSO solutions. The NMR constraints obtained were employed in the selection of starting conformations of the cyclic moiety of the analog. In particular, the diminished accessibility of the Asn5 NH proton to solvent and the close contact between Cpp1 and Cys6 C alpha H protons suggests a beta-turn conformation at the Phe3-Gln4 residues. Energy minimization was carried out both in the ECEPP/2 (rigid-valence geometry) and in the AMBER (flexible-valence geometry) force fields. Comparison of the experimental and calculated values of NMR characteristics has revealed that conformations containing type I, II, and III beta-turns at the Phe3-Gln4 residues are in reasonable agreement with the experimental data, with a dynamic equilibrium between the beta I (beta III) and beta II type structures of the cyclic part being the most probable. All of these conformations prefer the negative chirality of the disulfide bridge (theta 3 approximately -90 degrees). Five representative conformations were chosen for the acyclic tail: one with a beta I, one with a beta II'-turn at the Sar7-Arg8 residues, two extended-type conformations, and a conformation with a gamma-turn at Sar7. Because only high-energy extended conformations were in agreement with NMR data, it was concluded that the acyclic tail has considerable conformational flexibility in solution. The conformations obtained are discussed in terms of the structure-function relationship of the neurohypophyseal hormone analogs.     

Structural and biological effects of a beta2- or beta3-amino acid insertion in a peptide.

Molecular mechanics calculations on conformers of Ac-HGly-NHMe, Ac-beta2-HAla-NHMe and Ac-beta3-HAla-NHMe indicate that low-energy conformations of the beta-amino acids backbone, corresponding to gauche rotamers around the Calpha-Cbeta bond, may overlap canonical backbone conformers observed for alpha-amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)-beta2-HPhe and of Gly9 by HGly (R)-beta2-HAla or (S)-beta3-HAla. [(R)-beta2-HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)-beta3-HAla9]SP show a 30- to 50-fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a beta-amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)-beta3-HAla9]SP and [(R)-beta2-HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of beta2-HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a beta-amino acid could replace an alpha-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the alpha-amino acid.