13C- and 1H-nmr evidence for all-cis peptide bonds in cyclic tetrapeptide cyclo(L-Pro-Sar)2

Conformations of the cyclic tetrapeptide cyclo(L -Pro-Sar)₂ in solution were studied by ¹H- and ¹³C-nmr spectrometry and model building. The nmr data provide definite evidence that this cyclic peptide exists chiefly in two conformations, namely, a C₂-symmetric conformation and an asymmetric structure. The former was demonstrated to be predominant in polar solvents (100% in Me₂SO-d₆). This structure contains all cis-peptide bond linkages and all trans′ Pro C^α?CO bonds. It represents the first cyclic tetrapeptide in which all four peptide bonds have been found in the cis-conformation. As the polarity of the solvent decreases, the population of C₂-symmetric conformers decreases (88% in CD₃CN and 65% in CDCl₃). At the same time, a minor asymmetric conformer, characterized by cis-cis-cis-trans peptide bond sequences (two cis Sar-Pro bonds, one cis Pro-Sar bond, and one trans Pro-Sar bond), is seen to increase (9% in CD₃CN and 30% in CDCl₃). A proposed predominant conformation in solution for cyclo(L -Pro-Sar)₂ was compared with a crystal structure, as reported in an accompanying paper. Both structures show striking overall similarities.     

Nmr studies of the molecular conformations in the linear oligopeptides H-(L-Ala)n-L-Pro-OH

The molecular conformations of the linear oligopeptides H-(L -Ala)_n-L -Pro-OH, with n = 1,2 and 3, have been investigated. ¹³C nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala)_n-Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the cis:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carboxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from ¹H nmr studies. However, the cis:trans ratio of the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions, whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model consideration which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline, respectively.     

Cation binding cyclic peptides composed of imino acid residues

Cyclo(L -Pro-Sar)_n (n = 2–4) with moderate flexibility and hydrophobicity of molecular structure was synthesized, and the characteristics of these cyclic peptides and their metal complexes in acetonitrile were investigated in connection with the residual properties using ¹³C-nmr measurements. The cyclic tetrapeptide cyclo(L -Pro-Sar)₂ showed a sterically hindered phenomenon in acetonitrile in which the amide backbone adopted a cis-trans-cis-trans sequence. The cyclic hexapeptide cyclo(L -Pro-Sar)₃ existed as a mixture of several conformers whose interconversion is slow on the nmr time scale, including cis-cis-trans and/or cis-trans-trans arrangement of the Sar-Pro bond. Finally, it was demonstrated that the cyclic octapeptide cyclo(L -Pro-Sar)₄ behaved as a mixture of multiple conformers which allowed for cis-trans isomerism about the Pro-Sar peptide bond, of which 20–30% had the all-cis Sar-Pro bond isomer and the remaining 70–80% had one (or more) cis Sar-Pro bond isomer. ¹³C-nmr spectra also demonstrated that cyclo(L -Pro-Sar)_n (n = 3,4) formed a 1:1 ion complex whose conformation was characterized by an all-trans peptide bond in the presence of excess metal salt. Cation binding studies, using CD measurements, established that the ion selectivity of cyclo(L -Pro-Sar)₄ in acetonitrile decreased in the order, Ba²⁺ > Ca²⁺ > Na⁺ > Mg²⁺ > Li⁺.     

Conformational differences between cis and trans proline isomers of a peptide antigen representing the receptor binding domain of Pseudomonas aeruginosa as studied by 1H-nmr

A 17-residue disulfide-bridged peptide (PAK 128–144) corresponding to the C-terminus of Pseudomonas aeruginosa pilin strain K has been studied by one- and two-dimensional nmr techniques. This synthetic immunogen has been found to exist as two distinct conformations in solution, which have been demonstrated to arise as a result of the isomerization of the I¹³⁸-P¹³⁹ amide bond. The two isomers occur in the ratio of 3 : 1 trans to cis at 5°C. Sequential assignments for both forms have been accomplished through the use of nuclear Overhauser enhancement spectroscopy (NOESY) spectra and most side-chain resonances have been assigned using a combination of correlated spectroscopy, total correlated spectroscopy, and NOESY spectra. The presence of the cis isomer, which is considerably more predominant in the oxidized peptide, was confirmed by the observation of the characteristic NOEs between P¹³⁹ and the preceding residue. Further corroboration was given by the disappearance of the cis resonances in the spectrum of the P139A analogue of PAK 128–144. From observation of the differences in the chemical shifts and amide proton temperature coefficients of the two isomers, it is apparent that the two forms differ markedly in their solution conformation. The biological implications of the isomerization are discussed. © 1994 John Wiley & Sons, Inc.     

Conformational transitions in a Pro-Pro peptide on dissolution of single crystals

The peptide t-butyloxycarbonyl-α-aminoisobutyryl-L-prolyl-L-prolyl-N-methylamide has been shown to adopt an extended structure in the solid state. The Pro-Pro segment occurs in the poly-proline II conformation. On dissolution of single crystals at ～ 233°K, a single species corresponding to the all $\text{trans}$ peptide backbone is observed by 270 MHz ¹H NMR. On warming, $\text{trans}$ to $\text{cis}$ isomerization about the Pro-Pro bond is facilitated. Both $\text{cis'}$ (ψ ～?50°) and $\text{trans'}$ (ψ ～ 130°) rotamers about the Pro³ C^αCO bond are detectable in the Pro-Pro $\text{cis}$ conformer, at low temperature. These observations demonstrate unambiguously the large differences in the solid state and solution conformations of a Pro-Pro sequence.     

Steric effects of cis-trans isomerism on neighboring residues in proline oligopeptides: A 13C-nmr study of conformational heterogeneity in linear tripeptides

A series of proline-containing linear oligopeptides (4 dipeptides and 15 tripeptides) were synthesized and examined in aqueous and nonaqueous solutions using ¹³C-nmr spectroscopy. Spectra of linear tripeptides showing cis-trans isomerism about the X-Pro bond (X = Pro, Gly, and Ala) also show neighboring effects on the chemical shifts of residues both preceding and following the prolyl moiety. The extent of cis-trans isomerism observed about the X-Pro peptide bond correlates not only with the nature of X, but also depends on the size of the residue following proline; the larger substituents favor an increase in cis content about the X-Pro bond.     

β-Alanine containing cyclic peptides with turned structure: The “pseudo type II β-turn.” VI

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L -Pro-L -Phe-β-Ala)₂. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L -Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d₆ solutions as a mixture of conformational families. One of these corresponds to a C₂ symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ?,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.     

Conformational characteristics of polypeptides containing isolated L-proline residues with cis peptide bonds

The conformational characteristics of the peptide sequence X-l-Pro, where X  Gly or l-Ala and the peptide bond joining X and l-Pro is cis, are evaluated. Semi-empirical potential functions are used to estimate the contributions to the conformational energy made by the non-bonded van der Waals' and electrostatic interactions and the intrinsic torsional potentials about the NC^a and C^aC′ bonds. Rotations φ₁ and ψ₁ about the NC^a and C^aC′ bonds in residue X and rotation ψ₂ about the C^aC′ bond in l-Pro are permitted, while the angle of rotation φ₂ about the NC^a bond in l-Pro is fixed at 120 ° by the pyrrolidine ring. The presence of the cis peptide bond connecting X and l-Pro renders the backbone rotations φ₁, ψ₁ in X dependent upon the rotation ψ₂ about the C^aC′ bond in l-Pro. (Interdependence of rotations in neighboring residues joined by a cis peptide bond was previously observed in l-alanine oligomers.) The number of energetically allowed conformations for the Gly and l-Ala residues preceding a cis peptide bond l-Pro residue are found to be substantially reduced from those permitted when the peptide bond is trans or when l-Pro is replaced by an amino acid residue. On the other hand, ψ₂ = 100 to 160 ° (cis′) and 300 to 0 ° (trans′) are found to be the lowest energy conformations of the l-Pro residue irrespective of the cis or trans conformation of the X-l-Pro peptide bond.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Solvent and side-chain contributions to the two-cis ⇄ all-trans equilibria of cyclic hexapeptides,cyclo(Xxx-Pro-D-Yyy)2

Cyclic hexapeptides of the type cyclo(L -Xxx-L -Pro-D -Yyy)₂ or cyclo(L -Xxx-L -Pro-Gly)₂ exist in solution predominantly in two forms of C₂ average symmetry, one with all-trans peptide bonds and generally well-established conformation, and another with both Xxx-Pro peptide bonds cis. We have been measuring the thermodynamic parameters of this equilibrium using carbon and proton nmr spectroscopy. Data have been obtained for peptides in which Yyy = Gly, D -Ala, or D -Phe, and Xxx = Gly, L -Ala, L -Leu, and L -Val. In a given solvent, stability of the all-trans form decreases (ΔG⁰ increases) as Xxx is changed through the series Gly, L -Ala-, L -Leu, and L -Val, consistent with expected increasing repulsion between the Xxx side chain and the proline δ methylene across the trnas Xxx-Pro bond. Also, for a given set of side chains, the stability of the all-trnas form increases as the polarity of the solvent decreases, consistent with models in which all C?O and N? H groups are accessible for solvation in the two-cis form, but two C?O and two N? H groups are somewhat sequestered in the all-trans form. With the available data it is not possible to identify pure intramolecular (solvent-independent) or pure peptide-bond solvation (side chain-independent) terms in ΔH° or ΔS°, although trends are discernible.     

A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Thermodynamics of cyclophilin catalyzed peptidyl-prolyl isomerization by nmr spectroscopy

One-dimensional nmr exchange spectroscopy was carried out to determine thermodynamic parameters of cyclophilin-induced cis-trans isomerization of succinyl-Ala-Phe-Pro-Phe-p-nitroanilide. Rate measurements were possible at physiological temperatures. The k_c/K_m of rat cyclophilin was found to he 12.8 (±0.5) s^?1 μM^?1 at 37°C, intermediate to previously reported values that used a coupled enzyme assay extrapolated to this temperature. Activation energies (ΔG^≠) for the uncatalyzed and catalyzed reaction at 37°C were found to be 19.7 and 17.1 kcal/mol, respectively, and were primarily due to an enthalpic barrier. © 1994 John Wiley & Sons, Inc.     

Determination of the rates and barriers to conformational isomerization in the dipeptide L-pro-L-4hyp by direct 13C nmr thermal equilibration

The ¹³C nmr equilibration method lends itself as a tool for study of conformational rate processes involving aqueous media in conjunction with high activation barriers. This method is applied for measurement of kinetic and thermodynamic parameters of isomerism in the dipeptide L -Pro-L -4Hyp. The activation barrier for cis ? trans interconversion (ω 0° → 180°) is determined, ΔG^≠ = 22.3 kcal/mol. From low-temperature study, an upper limit ΔG^≠ < 9.7 kcal/mol is evaluated for cis′ ? trans′ rotation (ψ ?40° → 160°). These data are compared with computed values found in literature. The results are discussed in connection with the helix–coil transition of collagen involving Gly-L -Pro-L -4Hyp as nucleation sites.     

Conformational energy calculations on glycosylated turns in glycoproteins

Analysis of the amino acid sequence of glycoproteins has suggested the β-turn as a likely site of glycosylation in glycoproteins. According to this model, the peptide chain traverses the interior of a globular protein, reversing its direction at the protein surface, a likely point for the attachment of hydrophilic carbohydrate residues. In order to search for plausible conformations of glycosylated β-turns in asparagine-linked glycoproteins, we have adapted the conformational energy calculation method of Scheraga and coworkers for use in carbohydrates. The parameters for nonbonded and hydrogen-bonded interactions have been published, and electrostatic parameters are derived from a CNDO calculation on a model glycopeptide. Our results indicate that the orientation of the glycosyl amide bond having the amide proton nearly trans to the anomeric proton of the sugar has the lowest energy. Although CD and nmr experiments in our laboratory have consistently found this conformation, our calculations show the conformation having these two protons in a cis relationship to lie very close in energy. Calculations on the glycopeptide linkage model, α-N-acetyl, δ-N(2-acetamido-1,2-dideoxy-β-D -glucopyranosyl)-N′-methyl-L -asparaginyl amide show that several distinct geometries are allowed for glycosylated β-turns. For a type I β-turn, three conformations of the glycosylated side chain are found within 4 kcal of the minimum, while two conformations of the glycosylated side chain are allowed for a type II turn. The hydrogen-bonded C7 conformation is also allowed. Stereoviews of the low-energy conformations reveal no major hydrogen-bonding interaction between the peptide and sugar.     

A theoretical estimate of the energy barriers between stable conformations of the proline dimer.

Semi-empirical energy calculations for an internal Pro-Pro dimer are presented that take into account the nature of the flexibility of the proline ring due to its puckering. Calculations show that three stable conformations are available for the dimer: the cis (ω = 0°, ψ = 160°); the trans (ω = 180°, ψ = 160°, also referred to as trans′); and the cis′ (ω = 180°, ψ = ?40°) conformations. The best conformational pathways between these stable conformations are determined. Calculations also show that the barrier for cis′–trans′ conversion is of the same order of magnitude as that for cis–trans conversion.     

Conformational aspect of polypeptide structure. XLIV. Conformational transitions of poly(N-methyl-alanines) induced by trifluoroacetic acid

The synthesis of poly(N-methyl-L -alanine) and poly (N-methyl-DL -alanine) are described. The polymers were examined by 220 MHz high-resolution nuclear magnetic resonance (nmr) and circular dichroism (CD). The results demonstrate that poly(N-methyl-L -alanine) exists as an ordered helical structure with all the amide bonds in the trans configuration in appropriate solvents. As trifluoroacetic acid (TFA) is added to the solutions of the polymer in helix-supporting solvents, resonances corresponding to both trans and cis amide conformations of N-methyl, C-methyl, and α-CH are observed. The presence of both the trans and the cis peptide bonds in a polymer chain disrupts the ordered structures. Our conclusions from CD data are in agreement with the nmr results. Ultracentrifugation shows that degradation of the polymer chain does not occur during the TFA treatment.     

Cis-trans isomerization of the Epstein-Barr virus determinant peptide EENLLDFVRF after the DM1 TCR recognition of the HLA-B*4405/peptide complex

The Epstein–Barr virus determinant peptide EENLLDFVRF shows high immunogenicity when presented by HLA-B*4405 allotype. This fact is accompanied by a cis–trans isomerization of the Leu5-Asp6 peptide bond upon TCR binding of the pMHC complex. Molecular dynamics simulations of pMHC/TCR structures, with the EENLLDFVRF peptide in cis and trans conformations have been employed in order to examine the structure and dynamics of the pMHC complex with such an unusual conformation. The results, based on MM-PBSA free energy computations as well as buried surface area analysis and interactions at the pMHC/TCR interface, indicate that the TCR binds preferably the pMHC complex with the Leu5-Asp6 peptide bond in cis conformation. It is the first time that this notable conformational feature of T-cell epitope is investigated.     

Bioactive peptides: Solid state,solution and molecular dynamics studies of a cyclolinopeptide A-related cystinyl cyclopentapeptide

The conformational analysis of the disulphide cyclopeptide-related cyclolinopeptide A, has been carried out by solid state methods using x-ray diffraction techniques, in solution by nmr, CD, ir spectroscopies, and by molecular dynamics (MD) analysis. The structure of the monoclinic form, obtained from ethanol (a = 11.303(2) Å, b = 14.467(8) Å, c = 12.355(2) Å, β(°) = 109.40(1), space group P2₁, Z = 2) presents two transannular H bonds with the formation of one type VIa β-turn involving the C ? O of the urethane moiety and the Phe³ NH, and an intramolecular H bond between the C ? O of urethane group and the Phe⁴ NH. In the solid state all the peptide bonds are in the trans configuration with the exception of a cis peptide bond occurring between the Cys¹ and Pro² residues; the linkage S—S assumes right-handed chirality. The conformational study in solution by nmr spectroscopy indicates that the peptide is very flexible and that some conformer families are present at room temperature both in polar and apolar solvents. CD studies confirm that this cyclic system tends to give rise to a complex mixture of quasi-isoenergetic conformations, favored by the flexibility of the disulphide bridge and by the isomerism of the Xxx-Pro bond. MD studies carried out in vacuo and in solution shows that the structure determined by solid state represents a energy minimum. All hydrogen conds found in the crystalline state are correctly reproduced in vacuo and in solution simulations. © 1994 John Wiley & Sons, Inc.     

The X-Pro peptide bond as an nmr probe for conformational studies of flexible linear peptides

The equilibrium between the cis and trans forms of X-Pro peptide bonds can readily be measured in the ¹³C nmr spectra. In the present paper we investigate how observation of this equilibrium could be used as an nmr probe for conformational studies of flexible polypeptide chains. The experiments include studies by ¹³C nmr of a series of linear oligopeptides containing different X-L -Pro peptide bonds, with X = Gly, L -Ala, L -Leu, L -Phe, D -Ala, D -Leu, and D -Phe. Overall the study confirms that X-Pro peptide bonds can generally be useful as ¹³C nmr probes reporting the formation of nonrandom conformation in flexible polypeptide chains. It was found that the cis–trans equilibrium of X-Pro is greatly affected by the side chain of X and the configuration of the α-carbon atom of X. On the basis of these observations some general rules are suggested for a practical applications of the X-Pro nmr probes in conformational studies of polypeptide chains.