NMR and CD studies of triple-helical peptides.

Triple-helix formation of the peptide (Pro-Hyp-Gly)10 was monitored by nmr and CD spectroscopy. The two-dimensional nmr spectra indicated that the Gly C alpha H and Pro C delta H proton resonances shift upfield in going from the nonhelical to helical form, while hydroxy-proline resonances are unchanged. The integrated areas of the helical and nonhelical resonances could be monitored in the one-dimensional nmr spectrum, and indicate that in the (Pro-Hyp-Gly)10 about 90% of the residues are in a defined triple-helical conformation. The introduction of a glycine to alanine substitution or the deletion of a single hydroxyproline residue in the stable triple-helical peptide (Pro-Hyp-Gly)10 still allows trimers to be formed, but the trimers show a substantial loss of triple helix and decreased thermal stability compared with (Pro-Hyp-Gly)10. Two computer models were generated for the Gly----Ala peptide, one with the Ala side chains packed inside the helix and the other with the region containing the alanines forming a beta-bend that loops out from the helix. The nmr data is more consistent with the latter model.     

Depiction of the forces participating in the 2-O-sulfo-alpha-L-iduronic acid conformational preference in heparin sequences in aqueous solutions

2-O-Sulfo-alpha-l-iduronic acid (IdoA2S) is one of the main components of heparin, an anticoagulant and antithrombotic polysaccharide able to potentiate the inhibitory effect of antithrombin over plasma serine proteases. This monosaccharide unit adopts an equilibrium between chair (1C4) and skew-boat (2SO) forms as a function of heparin sequence size and composition. Although the prevalence of the 1C4 chair conformation in monosaccharides is understood, the reasons for the increase in 2SO contribution in the whole polysaccharide chain are still uncertain. In this context, 0.2 mus molecular dynamics simulations of IdoA2S-containing oligosaccharides indicated that stabilization due to intramolecular hydrogen bonds around IdoA2S is highly correlated (p0.001) with the expected conformational equilibrium for this residue in solution. This behavior explains the known effect of different heparin compositions, at the monosaccharide level, on IdoA2S conformation in biological solutions.     

Conformational analysis of dolastatin 10: An nmr and theoretical approach

A solution conformational analysis of dolastatin 10, a powerful antineoplaslic agent, has been carried out by means of nmr techniques and theoretical calculations. ¹H mono- and bidimensional nmr experiments, as well as ¹H-¹³C heterocorrelated spectra, have been performed on CD₂Cl₁₂ solutions. The most interesting nmr data is a huge shielding of the aCH (25) proton of the Dov residue, suggesting the presence of an interaction between the N-terminal and the aromatic C-terminal ends of the molecule. The possibility of a head-to-tail intermolecular association having been discarded, the presence of a series of preferred folded conformation has been hypothesized. Conformational theoretical analysis supports the nmr hypothesis of a folded peptide-like molecule, and a series of possible conformers in good agreement with the experimental data have been analyzed. © 1995 John Wiley & Sons, Inc.     

Polypeptide models of collagen. II. Solution properties of (Pro-Gly-Phe)n

The conformation of (Pro-Gly-Phe)_n in trifluoroethanol was investigated using CD, nmr and ir techniques. After making appropriate correction for the contribution of the phenylalanine chromophore to the observed CD spectra of the polytripeptide at several temperatures, it is found that (Pro-Gly-Phe)_n can exist in a partially triple-helical conformation in this solvent a t low temperatures. The nmr and ir data support this conclusion. In conjunction with recent theoretical sutdies, our data offer an explanation for the preferential occurrence of the Phe residue in position 2 of the tripeptide sequence Gly-R₂-R₃, in collagen.     

Conformational analysis of bacitracin A, a naturally occurring lariat

The proton nmr spectra of bacitracin A in H2O and DMSO-d6 have been assigned and the conformational behavior of the peptide in the two solvents has been compared. Although bacitracin A shows a conformational equilibrium between at least two conformations differing in the relative position of the cyclic and linear domains of the molecule, the spectra in water can be interpreted in terms of a preferred conformation in which the linear part is folded over the cyclic moiety and a turn is present around Ile(8)-DPhe(9).     

Conformational studies of the pentapeptides with weak adrenocorticotropin (ACTH) activities by means of nuclear magnetic resonance spectroscopy

The conformations of a pentapeptide L -His-L -Arg-L -Trp-Gly with weak adrenocorticotropin (ACTH) activity and its analogs, where each L -amino acid residue is substituted by D -residue, were investigated by means of proton and carbon-13 nmr spectroscopy on their DMSO-d₆ solutions. The spectra indicated the presence of slowly exchangeable conformation isomers for D -Phe and D -Arg analogs, due to steric hindrance around the arginine residue. The activation energy of the hindered rotation of the arginine side chain was estimated to be more than 19 ～ 20 kcal/mol. Spin-lattice relaxation times of carbon-13 nuclei also indicated slow segmental motion of the arginine side chain of the D analogs. An effect on proton chemical shifts by intermolecular electrostatic interaction between the arginine side chain and the C terminal carboxylic residue was observed. We did not observe, however, a direct correlation between pentapeptide activity and molecular conformation at this stage of the experiments.     

CD and nmr studies on the interaction of lithium ion with valinomycin and gramicidin-S

The conformation of the valinomycin–lithium complex has been studied using CD and nmr techniques. The lithium ion induced significant changes in the chemical shifts of the NH and C^αH protons, as well as in the CD spectra of valinomycin. From the analysis of the lithium ion titration data, it is concluded that valinomycin forms a 1:1 type weak complex with lithium, having a stability constant of 48 L mol^?1 at 25°C. This conformation is different from the familiar valinomycin–potassium complex. The nature of the interaction at low and high concentrations of lithium ions with valinomycin (ionophore) and gramicidin-S (nonionophore) has been compared. At high salt concentrations, there was a further change in the CD and nmr spectra of valinomycin, giving a second plateau region at > 3M of the salt. In the case of gramicidin-S, no significant changes in the nmr or CD spectra were observed in the lower concentration range corresponding to where changes were observed in the case of valinomycin. However, the addition of lithium salt at concentrations greater than 3M induced changes in both the CD and nmr spectra of gramicidin-S, and the titration graph of molar ellipticity versus concentration of lithium perchlorate gave a plateau region at concentrations greater than this. These results indicate that the effects of lithium at low and high concentrations are independent of each other. The conformational transitions at very high salt concentrations (denaturation) are more likely due to solvent structural perturbations rather than to the consequences of ion binding.     

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.     

Sub-angstrom conformational changes of a single molecule captured by AFM variance analysis

A system's equilibrium variance can be analyzed to probe its underlying dynamics at higher resolution. Here, using single-molecule atomic-force microscope techniques, we show how the variance in the length of a single dextran molecule can be used to establish thermodynamic equilibrium and to detect conformational changes not directly observable with other methods. Dextran is comprised of a chain of pyranose rings that each undergoes an Angstrom-scale transition from a chair to boat conformation under a stretching force. Our analysis of the variance of the molecule's fluctuations verifies equilibrium throughout the force-extension curve, consistent with the expected thermodynamic ensemble. This validates further analysis of the variance in the transition region, which reveals an intermediate conformation between the chair and the boat on the sub-Angstrom scale. Our test of thermal equilibrium as well as our variance analysis can be readily extended to a wide variety of molecules, including proteins.     

Conformation-based design of two cyclic physalaemin analogues

Two new cyclic analogues of physalaemin were designed on the basis of the conformation found in DMSO solution. Glp-Ala-cyclo(-Asp-Pro-Asn-Lys-)-Phe-Tyr-Gly-Leu-Met-NH2 (1) was synthesized by cyclization of physalaemin. In 2 the Asp residue was replaced by Glu. The linear analogue of 2 was synthesized by the solid phase method and subsequently cyclized. Two-dimensional nmr methods were employed to assign the proton and carbon resonances. Proton-proton distances were extracted from rotating frame nuclear Overhauser effect spectra and used as restraints in the molecular dynamics calculations. Analogue 1 was found to have a similar conformation as physalaemin, whereas 2 did not form intramolecular hydrogen bonds. The pharmacological evaluation revealed that both peptides have similar potencies as physalaemin in the dog carotid artery (NK-1 receptor). Therefore, the charged side chains of physalaemin appear not essential for NK-1 activation. However, the other tachykinin receptors show good sensitivity to the cyclic peptides. It is concluded that the replacement of a salt bridge by an amide bond connecting the side chains of natural residues might provide useful information about the biological significance of some charged side chains of neurokinins.     

Conformational analysis of CCK-B agonists using 1H-nmr and restrained molecular dynamics: Comparison of biologically active Boc-Trp-(N-Me)Nle-Asp-Phe-NH2 and inactive Boc-Trp-(N-Me)Phe-Asp-Phe-NH2

The tetrapeptide Boc-Trp-(N-Me)Nle-Asp-Phe-NH₂ is a potent CCK-B agonist. Replacement in this analogue of the norleucine residue by a phenylalanine, to yield Boc-Trp-(N-Me)Phe-Asp-Phe-NH₂, led to a 740-fold decrease in affinity whereas the same decrease in affinity was not observed in their nonmethylated counterparts. In order to ascertain the conformational preferences of these two N-methylated tetrapeptides, a study by two-dimensional (2D) nmr spectroscopy and molecular modeling was undertaken. The solution conformation of the two peptides was examined by ¹H-nmr in a d₆-DMSO/H₂O (80 : 20) mixture. A cis-trans equilibrium, induced by N-methylation, was observed for both analogues, and the proton spectra of the two retamers were fully characterized in each case. ¹H-¹H distance constraints, derived from 2D nuclear Overhauser effect spectroscopy and rotating frame nuclear Overhauser effect spectroscopy experiments, were used as inputs for subsequent restrained molecular dynamics simulations. Comparisons of the nmr and molecular modeling data point toward distinct conformational preferences for these two peptides with an opposite spatial orientation of the Trp residue, and could explain the large difference in their biological activities. Furthermore, the tridimensional structure of Boc-Trp-(N-Me)Nle-Asp-Phe-NH₂ could serve as a model for the design of nonpeptide CCK-B agonists. © 1994 John Wiley & Sons, Inc.     

XII. Conformational studies of histidine-containing peptides in solution

The spectroscopic properties (uv, CD, nmr) of histidine, glycylhistidine, histidylglycine, glycylhistidylglycine have been investigated in water and methanol in the temperature range 200–320 K in order to obtain information about their conformational equilibria. This analysis has been carried out for the different ionic forms of the compounds, in order to evaluate the influence of the ionization state of the carboxyl, histidyl, and amino groups on the rotamer distribution of the histidyl side chain (as evaluated from proton nmr analysis) and on the overall molecule (as judged from CD spectra). On the basis of certain approximations and from the temperature dependence of the proton nmr resonance, the thermodynamic parameters (ΔH° and ΔS°) characterizing the conformational equilibrium of the hystidyl side chain have been evaluated for the different structures and ionization states. Relatively large entropy differences between the rotamers are obtained in some cases. The data of the sidechain rotamer population, as determined by nmr, have been analytically correlated with the CD data, and in the case of hystidine and histidylglycine in basic solution, first-approximation values for the ellipticity of the single conformers have been evaluated. Finally, in the example of glycylhistidine and histidylglycine in basic solution, it is shown how the data obtained from the different experimental approaches (nmr and CD), as well as from theoretical energy calculations, converge to characterize the most stable conformation in solution.     

The conformational characteristics in solution of the synthetic cyclic hexapeptide 5L-ala-D-ala

An attempt to elucidate the solution conformation(s) of the synthetic cyclic hexapeptide 5L -ala·D-ala is described. Nuclear magnetic resonance (nmr) spectra are recorded for the purpose of measuring the vicinal coupling constant between the amide and α-protons in each residue and to observe the deuterium exchange rate and temperature dependence of the chemical shift of each amide proton. Low-energy cyclic conformations, whose individual residues are in conformations consistent with the observed amide to α-proton coupling constant, are searched for in an approximate theoretical treatment. The two lowest energy, all trans peptide bond conformations generated are distinguishable by the presence or absence of a single intramolecular hydrogen bond. The observed temperature independence of the chemical shift of one of the amide protons is consistent with the presence of a single intramolecular hydrogen bond, while the observation of similar deuterium exchange rates for each of the amide protons indicates their comparable availability to solvent. Consequently, it is concluded that 5L -ala·D-ala is in rapid equilibrium between conformations with and without a single internal hydrogen bond and possesses considerable conformational flexibility in solution.     

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.     

B3LYP/6-311++G* * study of structure and spin-spin coupling constant in heparin disaccharide

Structures of heparin disaccharide have been analyzed by DFT using the B3LYP/6-311++G( * *) method. The optimized geometries of two forms of this disaccharide, differing in the conformation ((1)C(4) and (2)S(0)) of the IdoA2S residue, confirmed considerable influences of the sulfate and the carboxylate groups upon the pyranose ring geometries. The computed energies showed that disaccharide having the (1)C(4) form of the IdoA2S residue is more stable than that with the (2)S(0) form. Interatomic distances, bond and torsion angles showed that interconversion of the IdoA2S residue results in geometry changes in the GlcN,6S residue as well. Three-bond proton-proton and proton-carbon spin-spin coupling constants computed for both forms agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium in disaccharide. Influences of the charged groups upon the magnitudes of spin-spin coupling constants are also discussed.     

Local control of peptide conformation: Stabilization of cis proline peptide bonds by aromatic proline interactions

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constants, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database. © 1998 John Wiley & Sons, Inc. Biopoly 45: 381–394, 1998     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.     

Solution conformations of two flexible cyclic pentapeptides: cyclo(Gly-Pro-D-Phe-Gly-Ala) and cyclo(Gly-Pro-D-Phe-Gly-Val).

In an effort to explore the residue preferences in three-residue reverse turns (so-called gamma-turns), two cyclic pentapeptides--cyclo(Gly1-Pro2-D-Phe3-Gly4-Ala5) (I) and cyclo(Gly1-Pro2-D-Phe3-Gly4-Val5) (II)--have been synthesized and analyzed by nmr. It was anticipated that the Gly-Pro-D-Phe-Gly portions of these molecules would favor a beta-turn conformation, leaving the remainder of the molecule to adopt a gamma turn, as seen in several previously studied model cyclic pentapeptides. The nmr data for both peptides in CDCl3 (5% DMSO-d6) and in neat DMSO-d6 indicate that the most populated conformation contains a distorted beta turn around Pro2-D-Phe3, which includes a gamma turn around D-Phe3. The distortion in the beta turn does not impede the formation of an inverse gamma turn around residue 5, and indeed, this conformation is observed in both peptides. Both the alanine and the bulkier valine residues are therefore found to be compatible with an inverse gamma turn. Molecular dynamics simulations on the title peptides are reported in the following paper. These simulations indicate that there is conformational flexibility around the D-Phe3-Gly4 peptide bond, which enables the formation of the gamma turn around D-Phe3. The third paper in this series explores the impact of a micellar environment on conformational equilibria in II.     

Nmr studies of aqueous solutions of tetra and branched peptides. I. Sequence determination of amino-acid residues and the assignment of peptide hydrogen signals.

Sequential determination of glycyl residues (and in several cases different amino-acid residues) in tetra and branched peptides using the nmr technique is reported. The method is based on changes in the nmr spectra of (1) the peptide hydrogens of the different residues and (2) the methylene groups of the glycyl residues, as a result of increasing the rate of the base-catalyzed exchange reaction of the peptide hydrogens. Hence, the spectral changes are pH dependent. However, the exact pH dependence is a function of the location of the residue in the peptide molecule. Thus, it is possible to determine the sequence of the amino-acid residues by studying the changes in the spectra with pH. For peptide molecules of known sequences, the above method can be used for unequivocal assignment of the peptide hydrogen signals.     

Experimental chiroptical verification of linkage flexibility in methyl 3-O-(α-D-mannopyranosyl)-α-D-mannopyranoside

Vacuum UV CD spectra of methyl 3-O-(α-D -mannopyranosyl)-α-D -mannopyranoside in D₂O and as a cast film were obtained in the 145–200 nM region. The disaccharide solution CD per residue is nearly identical to that of the monosaccharide solution CD, and to the monosaccharide film CD. Conversely, the disaccharide film spectrum exhibits a strong positive CD linkage contribution in the 160–170 nm range, which is consistent with the known crystal conformation under the aegis of previously determined sector rules. The close similarity between the monosaccharide and disaccharide solution spectra, therefore, reflects conformational averaging in which the net linkage contribution is approximately zero. The present observation of significant solution linkage flexibility confirms previous conclusions based on optical rotation, as well as conclusions of others based on nmr data. Moreover, when combined with those earlier results, the present work demonstrates the population of at least three distinct potential energy wells on the disaccharide ϕ, ψ potential energy surface. © 1996 John Wiley & Sons, Inc.