Vibrational analysis of peptides,polypeptides, and proteins. V. Normal vibrations of β-turns

The normal modes have been calculated for β-turns of types I, II, III, I′, II′, and III′. The complete set of frequencies is given for the first three structures; only the amide I, II, and III modes are given for the latter three structures. Calculations have been done for structures with standard dihedral angles, as well as for structures whose dihedral angles differ from these by amounts found in protein structures. The force field was that refined in our previous work on polypeptides. Transition dipole coupling was included, and is crucial to predicting frequency splittings in the amide I and amide II modes. The results show that in the amide I region, β-turn frequencies can overlap with those of the α-helix and β-sheet structures, and therefore caution must be exercised in the interpretation of protein bands in this region. The amide III modes of β-turns are predicted at significantly higher frequencies than those of α-helix and β-sheet structures, and this region therefore provides the best possibility of identifying β-turn structures. Amide V frequencies of β-turns may also be distinctive for such structures.     

Vibrational analysis of peptides, polypeptides and proteins. XXVII. Structure of gramicidin S from normal mode analyses of low-energy conformations

Normal mode calculations have been carried out on three low-energy structures of gramicidin S obtained from conformational energy calculations. When the results on the amide modes are compared with observed bands in the infrared and Raman spectra of crystalline gramicidin S and its N-deuterated derivative, one of the structures is clearly disfavored. Of the other two, one is slightly favored, and it corresponds to the lowest-energy structure obtained from the energy calculations. Spectra from solutions in DMSO and CH3 OH suggest that the molecular conformation is essentially retained in these solvents.     

Evidence for a gamma-turn motif in antifreeze glycopeptides.

Knowledge of the secondary structure of antifreeze peptides (AFPs) and glycopeptides (AFGPs) is crucial to understanding the mechanism by which these molecules inhibit ice crystal growth. A polyproline type II helix is perhaps the most widely accepted conformation for active AFGPs; however, random coil and alpha-helix conformations have also been proposed. In this report we present vibrational spectroscopic evidence that the conformation of AFGPs in solution is not random, not alpha-helical, and not polyproline type II. Comparison of AFGP amide vibrational frequencies with those observed and calculated for beta and gamma-turns in other peptides strongly suggests that AFGPs contain substantial turn structure. Computer-generated molecular models were utilized to compare gamma-turn, beta-turn, and polyproline II structures. The gamma-turn motif is consistent with observed amide frequencies and results in a molecule with planar symmetry with respect to the disaccharides. This intriguing conformation may provide new insight into the unusual properties of AFGPs.     

Quantitative evaluation of gamma-turn conformation in proline-containing peptides using 13C N.M.R.

The dependence of the 13C shift difference of proline carbons C beta and C gamma on the dihedral angle psi has been studied using the model peptide acetyl-D-proline N-methylamide. The shift difference delta beta gamma is shown to be correlated with the percent cis isomer about the acetylproline bond, both factors depending strongly on the degree of intermolecular hydrogen bonding. Both the fraction of trans peptide bond and the fractional gamma-turn conformation increase as the sample concentration is decreased in CDCl3. delta beta gamma values have been used to evaluate the fractional gamma-turn probabilities in a number of cyclic and linear peptides including thyrotropin releasing factor and bradykinin. Using this parameter, it is concluded that in bradykinin the gamma-turn probability is low in D2O and not strongly temperature dependent. In contrast, studies of a model peptide for the portion of bradykinin believed to adopt a gamma-turn conformation are consistent with an increased gamma-turn probability inless polar solvents. Data for X-Pro-Y peptides (Y = imino acid) indicate significantly reduced values of delta beta gamma, and this appears to be a useful basis for assigning the Pro C beta resonances corresponding to this sequence.     

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.     

Backbone modifications in cyclic peptides. Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess beta- and gamma-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Pro psi[CH2S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K2CO3 and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by 1H and 13C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a gamma-turn intramolecular hydrogen bond in the all-amide structure. In CDCl3, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining beta-turn hydrogen bond was weaker according to delta delta/delta TNH measurements. In DMSO-d6, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly interconverted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. 13C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly1-Pro2 position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

Vibrational analysis of peptides,polypeptides, and proteins. VI. Assignment of β-turn modes in insulin and other proteins

The normal modes have been calculated for structures having the dihedral angles of the four β-turns of insulin. Frequencies are predicted in the amide I region near 1652 and 1680 cm^?1. The former overlaps the α-helix band at 1658 cm^?1 in the Raman spectrum, while the latter accounts for the hitherto unassignable band at 1681 cm^?1. Calculated amide III frequencies extend above 1300 cm^?1, providing a compelling assignment of the 1303-cm^?1 band in insulin and similar bands in other globular proteins.     

Vibrational analysis of peptides,polypeptides, and proteins. XXI. β-Calcium-poly(L-glutamate)

The observed Raman and ir spectra of Ca-poly(L -glutamate) in the β conformation have been analyzed by means of a normal mode calculation. The force field for the main chain was transferred without refinement from β-poly(L -alanine), yet it provides a good prediction of the observed bands and, in particular, explains subtle differences in the spectra of these two β-sheet structures. Main- and side-chain modes are well characterized, and the dependence of the amide III frequency on side-chain composition is again demonstrated.     

Normal mode spectrum of the parallel-chain β-sheet

Normal mode calculations have been carried out for parallel-chain β-sheet structures. These include the parallel-chain pleated sheet of poly(L -alanine) and the parallel-chain rippled sheet of polyglycine. Dipole derivative coupling has been included for amide I and II modes, and the effects of parallel-sheet and antiparallel-sheet arrangements of varying separation have been examined for the poly(L -alanine) case. Some amide and nonamide modes are distinctly different from their antiparallel-chain counterparts, thus providing a basis for distinguishing between such structures from their ir and Raman spectra. As in our previous studies, these results emphasize the need for both kinds of spectral data in order to draw definitive conclusions about conformation.     

Unique molecular conformation of aureobasidin A, a highly amide N-methylated cyclic depsipeptide with potent antifungal activity: X-ray crystal structure and molecular modeling studies.

A structural feature of aureobasidins, cyclic depsipeptide antibiotics produced by Aureobasidium pullulans R106, is the N-methylation of four out of seven amide bonds. In order to investigate possible relationship between the molecular conformation and the amide N-methylation, aureobasidin A (AbA), which exhibits the potent antifungal activity, was subjected to X-ray crystal analysis. The crystal, recrystallized from ether (orthorhombic, space group P2(1)2(1)2(1), a = 21.643 (3) A, b = 49.865(10) A, c = 12.427 (1) A, z= 8), contained two independent conformers per asymmetric unit and they took on a similar arrowhead-like conformation. The conformation consisted of three secondary structures of antiparallel beta-sheet, and beta- and gamma-turns, and was stabilized by three intramolecular and transannular N-H O=C hydrogen bonds. The beta-hydroxy-N-methyl-l-valine residue, which is indispensable for its bioactivity, was located at the tip of the corner. Since a nearly identical conformation has been observed for aureobasidin E, a related cyclic depsipeptide, this arrowhead-like conformation may be energetically stable and important for biological activity. The contribution of the amide N-methylation to the conformation was investigated by model building and energy calculations. The energy-minimizations of AbA analogs, in which some (one to four) of four N-methylated amide bonds were replaced with usual amide bond, led to some conformers which are fairly different from the arrowhead form of AbA, although they are stabilized by three intramolecular N-H...O=C hydrogen bonds. This result explains the reason why four out of the seven amide bonds have to be methylated to manifest biological activity, i.e. the high N-methylation of aureobasidin is necessary to form only one well-defined conformation.     

Vibrational analysis of peptides, polypeptides, and proteins. XXXII. alpha-Poly(L-glutamic acid)

The Raman and ir spectra of α-helical poly(L -glutamic acid) have been assigned on the basis of a normal mode calculation for this structure. The force field was based on our previously refined main-chain force constants for α-poly(L -alanine) and side-chain force constants for β-calcium–poly(L -glutamate). Despite the identical backbone α-helical structures, significantly different frequencies are calculated, and observed, in the amide III and backbone stretch regions of α-poly(L -glutamic acid), as compared with α-poly(L -alanine). This clearly demonstrates the influence of side-chain structure on mainchain vibrational modes.     

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.     

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.     

VCD Robustness of the Amide‐I and Amide‐II Vibrational Modes of Small Peptide Models

The rotational strengths and the robustness values of amide‐I and amide‐II vibrational modes of For(AA)_nNHMe (where AA is Val, Asn, Asp, or Cys, n = 1–5 for Val and Asn; n = 1 for Asp and Cys) model peptides with α‐helix and β‐sheet backbone conformations were computed by density functional methods. The robustness results verify empirical rules drawn from experiments and from computed rotational strengths linking amide‐I and amide‐II patterns in the vibrational circular dichroism (VCD) spectra of peptides with their backbone structures. For peptides with at least three residues (n ≥ 3) these characteristic patterns from coupled amide vibrational modes have robust signatures. For shorter peptide models many vibrational modes are nonrobust, and the robust modes can be dependent on the residues or on their side chain conformations in addition to backbone conformations. These robust VCD bands, however, provide information for the detailed structural analysis of these smaller systems. Chirality 27:625–634, 2015 © 2015 Wiley Periodicals, Inc.     

NMR studies on turn mimetic analogs derived from melanocyte-stimulating hormones

Oligomers with alpha-aminooxy acids are reported to form very stable turn and helix structures, and they are supposed to be useful peptidomimetics for drug design. A recent report suggested that homochiral oxa-peptides form a strong eight-member-ring structure by a hydrogen bond between adjacent aminooxy-acid residues in a CDCl3 solution. In order to design an alpha-MSH analog with a stable turn conformation, we synthesized four tetramers and one pentamer, based on alpha-MSH sequence, and determined the solution structures of the molecules by two-dimensional NMR spectroscopy and simulated annealing calculations. The solution conformations of the three peptidomimetic molecules (TLV, TDV, and TLL) in DMSO-d6 contain a stable 7-membered-ring structure that is similar to a gamma-turn in normal peptides. Newly-designed tetramer TDF and pentamer PDF have a ball-type rigid structure that is induced by strong hydrogen bonds between adjacent amide protons and carbonyl oxygens. In conclusion, the aminooxy acids, easily prepared from natural or unnatural amino acids, can be employed to prepare peptidomimetic analogues with well-defined turn structures for pharmaceutical interest.     

Conformational aspects of beta-trypsin interactions with substrates and pancreatic trypsin inhibitor. II. Structure of tetrahedral adducts and acylenzyme

Theoretical conformational analysis of the tetrahedral complexes of trypsin with the N-acetyl-L-lysine methyl amide, which are formed at the acylation and the deacylation stages of the catalytical act has been carried out. The lowest energy conformations are shown to be productive ones. All favorable structures of N-acetyl-L-lysyl-trypsin and N-acetyl-L-arginyl-trypsin acylenzymes have been analysed. The global conformations of both complexes are found to be very similar with the structures providing for a transition to the second tetrahedral state. Conformations of the nonbonded, tetrahedral and acyl complexes with N-acetyl-L-lysine methyl amide are compared and the differences in orientation of atomic groups participating in the catalysis are revealed. All changes of optimal structures of the complexes indispensable for the catalytical process are shown to proceed in a spontaneous way without introduction of any intramolecular strain.     

Nuclear magnetic resonance studies on a cyclic dodecapeptide analogue of a repeat hexapeptide of tropoelastin: evaluation of secondary structure.

The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems.     

Depsipeptide analogues of elastin repeating sequences: conformational analysis

In this work the effect of elimination of a specific hydrogen bond on the conformation of the repeating peptides of elastin was studied. These repeating sequences are the pentapeptide Val-Pro-Gly-Val-Gly and the hexapeptide Val-Ala-Pro-Gly-Val-Gly. These sequences have been proposed to occur in a beta-turn conformation with a hydrogen bond involving the amide NH of the internal valine residue and the carbonyl oxygen of the residue preceding proline. In the depsipeptide analogues studied in this work, this 4-1 beta-turn hydrogen bond cannot occur. We studied the depsipeptide sequences Val-Pro-Gly-Hiv-Gly and Val-Ala-Pro-Gly-Hiv-Gly (Hiv denotes S-alpha-hydroxyisovaleric acid, the hydroxy acid analogue of valine), as well as the peptide sequences Val-Pro-Gly-Val-Gly and Val-Ala-Pro-Gly-Val-Gly. Compounds studied included sequences with the Boc and benzyl ester protecting groups, derivatives with the acetyl and N-methylamide end groups and polymers of the above sequences. Our conclusions are based on a comparison of depsipeptides with analogous peptides. Conformational analysis was carried out by nmr, CD, and ir spectroscopy. We propose that in the repeating sequences of elastin an equilibrium exists between a gamma-turn structure and a beta-turn structure in the Pro-Gly segment resulting in a structure that combines flexibility with strong conformational preferences. The C7 involves the amide NH of the internal glycine and the carbonyl oxygen of the residue preceding proline. In the N-methylamide derivatives a similar equilibrium exists in the Gly-Val-Gly segment. In the depsipeptides the beta-turn cannot occur and only the gamma-turn is seen. In the polydepsipeptides the major conformational feature is a type I beta-turn involving Gly5 NH and Pro CO.     

Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton n.m.r. spectrum

With its simple glycotripeptide repeating structure the antifreeze glycoprotein of polar fish may be an especially simple conformational mode for mucin glycoproteins with similar but more complex structures. The fully assigned proton n.m.r. spectrum confirms the anomeric configurations of the hexapyranosidic sugars of the side chains and the coupling constants of the alpha GalNAc and the beta Gal residues show both to be in the expected 4C1 chair conformation. The assignment of a single resonance for each proton of the (Ala-Thr-Ala)n repeat unit coupled with the observation of long range nuclear Overhauser effects (n.O.e.) implies a three-fold repeating conformation. The resonances of the two alanines are distinct and can be assigned to their correct positions in the peptide sequence by n.O.e. observed at the amide proton resonances on saturation of the alpha proton signals. The amide proton coupling constants of all three peptide residues are similar and imply a limited range of peptide backbone torsion angles, phi CN. The large n.O.e. which has been observed between the amide proton and the alpha proton of the residue preceding it in the sequence implies large positive values for the peptide dihedral angle, psi CC. Limits are placed on possible values of side chain dihedral angles by the observation of the coupling constant between the alpha and beta protons of the threonyl residue. The observation of n.O.e. between the anomeric proton of GalNAc and the threonyl side chain protons gives information on the conformation of the alpha glycosidic linkage between the disaccharide and the peptide. n.O.e. observed between the protons of the beta glycosidic linkage indicates the conformation of the disaccharide and the large amide proton coupling constant of the GalNAc residue shows a trans proton relationship. The spectroscopically derived data have been combined with conformational energy calculations to give a conformational model for antifreeze glycoprotein in which the hydrophobic surfaces of the disaccharide side chains are wrapped closely against a three-fold left handed helical peptide backbone. The hydrophilic sides of the disaccharides are aligned so that they may bind to the ice crystal face, which is perpendicular to the fast growth axis inhibiting normal crystal growth.     

Conformational study of Met-enkephalin based on the ECEPP force fields

We report a computational study of the small peptide Met-enkephalin based on the ECEPP/2 and ECEPP/3 force fields using the basin paving method. We have located a new global minimum when using the ECEPP/3 force field with peptide angles omega fixed at 180 degrees. With this new result, we can conclude that the lowest energy configurations of Met-enkephalin predicted based on all four versions of ECEPP have a classic gamma-turn centered at residue Gly3 and a beta-turn at residues Gly3-Phe4. However, minor differences between the structures also exist.