Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Conformational analysis of biologically active thyroliberin analogs by two-dimensional NMR spectroscopy]

Preferable conformations of thyrotropin-releasing hormone (TRH, Glp-His-Pro-NH2) and its analogues Glp-Glu(R)-Pro-NH2 (R = NHCH(CH3)CH2Ar), Glp-Gln-Abu-NH2, Dho-Gln-Abu-NH2 in DMSO solution are determined using two-dimensional 1H NMR spectroscopy (delta-J-correlated, COSY and NOESY). Torsion angles psi i and chi i for every amino acid were calculated on the basis of the spin-spin coupling constants 3JNH-C alpha H and 3JC alpha H-C beta H values. The NOESY data were used for selecting the peptide conformations realized in solution. Distances between protons interacting by the dipole mechanism (d-contacts) were calculated using NOE values. These experiments allow one to estimate the torsion angles psi (between C alpha H-CO). TRH has an intramolecular H-bond between NH2-protons and His carbonyl with the torsion angles omega 3 = 180 degrees and psi 3 = 0 degrees. It is formation of this H-bond that apparently promotes the domination of the trans configuration of the His-Pro peptide bond. An intramolecular NH2-C alpha CO (Glp) H-bonding is revealed in other investigated compounds. It is known that a similar conformation of the TRH is realized in the course of its interaction with receptor.     

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.     

13C multiplet nuclear magnetic resonance relaxation-derived ring puckering and backbone dynamics in proline-containing glycine-based peptides.

13CH2-multiplet nuclear magnetic resonance relaxation studies on proline (P)-containing glycine (G)-based peptides, GP, PG, GPG, PGG, and GPGG, provided numerous dipolar auto- and cross-correlation times for various motional model analyses of backbone and proline-ring bond rotations. Molecular dynamics simulations and bond rotation energy profiles were calculated to assess which motions could contribute most to observed relaxation phenomena. Results indicate that proline restricts backbone psi 1, psi 2, and phi 2 motions by 50% relative to those found for a polyglycine control peptide. psi 1 rotations are more restricted in the trans-proline isomer state than in the cis form. A two-state jump model best approximates proline ring puckering which in water could occur either by the C gamma endo-exo or by the C2 interconversion mechanism. The temperature dependence (5 degrees to 75 degrees C) of C beta, and C gamma, and C delta angular changes is rather flat, suggesting a near zero enthalpic contribution to the ring puckering process. In lower dielectric solvents, dimethylsulfoxide and methanol, which may mimic the hydrophobic environment within a protein, the endo-exo mechanism is preferred.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides. I. Proton shifts in the ribose ring of pyrimidine nucleosides as a function of the torsion angle about the glycosyl bond

Molecular orbital computations are performed on the different contributions to the variation of the chemical shifts of the non-exchangeable protons of the ribose ring in pyrimidine nucleosides as a function of the torsion angle X_CN about the glycosyl bond. They show that the ring current effects are negligible, that the contribution of the atomic diamagnetic anisotropy is important for protons which come at very short distances to the anisotropic group (C₂ = 0₂) and that the polarization effect may have a determining influence on the sign of the variation of the chemical shift. The theoretical results are discussed in relation to the experimental findings on the differences between the chemical shifts of the ribose protons of pyrimidine nucleosides methylated at C₅ and C₆.     

Simplification and spin-spin analysis of the side chain proton magnetic resonance spectrum of the decapeptide gramicidin S using difference scalar decoupling and biosynthesis of specifically deuterated analogs.

Biosynthesis of specifically deuterated molecules and difference scalar decoupling permitted an analysis of all C alpha-C beta spin systems of gramicidin S. Proof is presented that proton magnetic resonance spectra obtained by difference scalar decoupling yield not only spectral assignments and simplification but also accurate chemicals shifts and scalar coupling constants. The variations in (3J alpha beta) and in proton chemical shifts at temperatures over the range of -54 degrees -+66 degrees C are consistent with the internal rotation around the C alpha-C beta bonds of Val1, Orn2, Leu3, and Phe4 residues discovered using carbon 13 spectroscopy. The value (3J alpha beta) = 1.5 Hz for the proline residue is consistent with there being only one C alpha-C beta conformer. This is supported by the small temperature dependence of (3J alpha beta). However, it cannot be rigorously excluded that oscillation between a major and a minor C alpha-C beta conformation occurs for proline.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides. II. Proton shifts in the ribose ring of purine nucleosides as a function of the torsion angle about the glycosyl bond

The variations of the ring current, the local diamagnetic susceptibility anisotropy and the polarization contributions to the chemical shift of the non exchangeable protons of the ribose ring of purine nucleosides are computed as a function of the torsion angle about the glycosyl bond, χ_CN. The results show that the ring current effect is relatively more important in the purines than in the pyrimidines. In addition, N₃ of purines has a local magnetic anisotropy effect similar to the one of the carbonyl group C₂O₂ of pyrimidine nucleosides. The experimental differences between the chemical shift of the ribose protons of purine nucleosides and of 8 substituted derivatives are discussed in relation to the theoretical variations.     

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.     

Persistence of the alpha-helix stop signal in the S-peptide in trifluoroethanol solutions

alpha-Helix formation in the S-peptide (residues 1-19 of ribonuclease A) was studied in detail by use of two-dimensional 1H nuclear magnetic resonance to monitor the effects of 2,2,2-trifluoroethanol (TFE) at 0 degrees C and pH* 2.07. TFE stabilizes the S-peptide alpha-helix. Helix formation by a particular amino acid was monitored by the chemical shifts of the C alpha, C beta, and C gamma protons while increasing the concentration of TFE: large changes in chemical shift of a particular residue indicate that it is induced to go helical, whereas small chemical shift changes indicate little helix formation. Residues Thr-3 to Met-13 undergo chemical shift changes consistent with helix formation, whereas the other residues do not. Earlier work [Kim, P. S., & Baldwin, R. L. (1984) Nature 307, 329-334] reported that residues Thr-3 to His-12 become helical in aqueous solution. The existence of a "helix stop signal" was inferred from this behavior. We thus conclude that this helix stop signal persists in TFE solutions.     

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.     

Two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin in solution

The solution conformation of [D-Ala2]-leucine enkephalin in its zwitterionic form in DMSO-d6 has been monitored by one- and two-dimensional proton magnetic resonance spectroscopy at 500 MHz. The resonances from the labile amide protons and the nonlabile protons have been assigned from the shift correlated spectroscopy. The chemical shift of the amide and C-alpha protons are found to vary with temperature but in opposite directions, except the C-alpha proton of the terminal tyrosine residue. This behavior has been explained by the shifting of equilibrium between the zwitterionic and neutral forms of the [D-Ala2]-leucine enkephalin and probably conformational changes accompanying temperature variation. The low values of the temperature coefficients of leucine and glycine amide protons indicate that these protons are either intramolecularly hydrogen bonded or solvent shielded. The observation of sequential cross peaks in the nuclear Overhauser effect spectra obtained at various mixing times, tau m (200-900 ms), indicate an extended backbone, which does not corroborate with the presence of a folded structure, i.e., beta-bend type structure. The estimate of interproton distances in conjunction with the low values of temperature coefficients of the leucine and glycine amide protons and vicinal coupling constants 3JHN-C alpha H have been rationalized by the predominance of two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin. The gamma-bend around the D-Ala residue has phi = 80 degrees and psi = 270 degrees, while the one around Phe it has phi = 285 degrees and psi = 90 degrees.     

SMS 201-995, a very potent analogue of somatostatin. Assignment of the 1H 500 MHz n.m.r. spectra and conformational analysis in aqueous solution

The conformations of a cyclic analogue of somatostatin, SMS 201-995, have been studied by n.m.r. spectroscopy at 500 MHz in aqueous solution. Assignments were made by use of 2D-correlated methods, especially by detecting long-range connectivities in order to identify the aromic amino-acid and long-range couplings between alpha protons of consecutive residues. Measurements of temperature coefficients of amide protons and of NH-C alpha H coupling constants enabled us to conclude that in water the molecule is rather flexible, with no evidence for a beta turn structure involving Thr6. An equilibrium involving two gamma turn conformations stabilized respectively by Cys2-D-Trp4 and Phe3-Lys5 hydrogen bonds, is responsible for the large upfield shift observed for the Lys5 gamma protons and is compatible with the measured JNH-C alpha H coupling constants.     

Quantitative determination of the conformation of ATP in aqueous solution using the lanthanide cations as nuclear-magnetic-resonance probes.

Chemical shift perturbations of the eight 1H resonances and of the three 31P resonances in the nuclear magnetic resonance spectra of ATP in 2H2O, pH 6.0, have been induced by specifically bound lanthanide cations Ln3+ (Ln = Pr, Nd, Eu, Yb). After separation of contact (through bond) perturbations the resultant through-space shifts, which are found to have axial symmetry, are used in an analysis of the conformation of the Ln3+ -ATP complex. A computer program was used to search for the conformations of the molecule which fit the nuclear magnetic resonance data. The "best" solutions obtained represent a small closely interrelated family of conformations. Effects of the cation Gd3+ on the longitudinal relaxation rates of five of the protons of ATP were also measured and used to confirm the conformational family. One of these conformations corresponds closely to one of the crystal structure forms, with an anti arrangement of the base-ribose unit and and a right-hand helical phosphate chain folded towards the adenine part of the molecule. The lanthanide ion binds predominantly to the beta and gamma phosphates and does not interact with the purine ring, these two centres being separated by at least one water molecule.     

Conformation and mobility of tyrosine side chain in tetrapeptides. Specific effects of cis- and trans-proline in Tyr-Pro- and Pro-Tyr-segments

We examined the properties of tyrosine in four free tetrapeptides: Ala-Ala-Tyr-Ala (AATA), Ala-Pro-Tyr-Ala (APTA), Ala-Tyr-Ala-Ala (ATAA) and Ala-Tyr-Pro-Ala (ATPA) by CD, n.m.r. and energy calculations. Experimental data (the aromatic 1Lb signal, rotamer populations around the C alpha-C beta bond (chi 1), rotations around C beta-C gamma(chi 2), chemical shifts of ortho- and meta-protons in the phenolic ring (in aqueous and Me2SO solutions), NH proton temperature coefficients and vicinal coupling constants 3JNH-C alpha H in the backbone (Me2SO solution) were compared with calculated minimum energy conformations. We find qualitative agreement between the results of the different techniques with respect to global tendencies of conformational behaviour: we present experimental evidence showing that the presence of proline in the sequence has a more pronounced effect on the side chain organization of the residues preceding it than on one succeeding it. This steric influence of proline on its immediate neighbor is even stronger in the cis isomer than in the more common trans isomer. The strong preference for Rotamer II (chi 1 = 180 degrees) over Rotamer I (chi 1 = -60 degrees) in ATPA (cis-form) concomitant with a noticeable deviation of chi 2 is a striking example.     

Ab-inito quantum mechanical calculations of NMR chemical shifts in nucleic acids constituents. II. Conformational dependence of the 1H and 13C chemical shifts in the ribose

The magnetic shielding constant of the different 13C and 1H nuclei of a deoxyribose are calculated for the C2' endo and C3' endo puckerings of the furanose ring as a function of the conformation about the C4'C5' bond. For the carbons the calculated variations are of several ppm, the C3' endo puckering corresponding in most cases to a larger shielding than the C2' endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose-3' and 5' phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides. The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

13C NMR Chemical Shifts of the Triclinic and Monoclinic Crystal forms of Valinomycin

Two different crystalline polymorphs of valinomycin, the triclinic and monoclinic forms, have been studied by high resolution, solid state (13)C CP-MAS NMR spectroscopy. Although the two polymorphs of the crystal are remarkably similar, there are distinct differences in the isotropic chemical shifts between the two spectra. For the triclinic form, the carbon chemical shift tensor components for the alpha carbons adjacent to oxygen in the lactic acid and hydroxyisovaleric acid residues and the ester carbonyls of the valine residue were obtained using the FIREMAT experiment. From the measured components, it was found that the behavior of the isotropic chemical shift, delta(iso), for valine residue ester carbonyl carbons is predominately influenced by the intermediate component, delta(22). Additionally it was found that the smallest shift component, delta(33), for the L -lactic acid ( L -Lac) and D -alpha-hydroxyisovaleric acid ( D -Hyi) C(alpha)-O carbon was significantly displaced depending upon the nature of individual amino acid residues, and it is the delta(33) component that governs the behavior of delta(iso) in these alpha carbons.     

Ab-inito Quantum Mechanical Calculations of NMR Chemical Shifts in Nucleic Acids Constituents II Conformational Dependence of the lH and 13C Chemical Shifts in the Ribose

Abstract

The magnetic shielding constant of the different ¹³C and ¹³H nuclei of a deoxyribose are calculated for the C2′ endo and C3′ endo puckerings of the furanose ring as a function of the conformation about the C4′C5′ bond. For the carbons the calculated variations are of several ppm, the C3′ endo puckering corresponding in most cases to a larger shielding than the C2′ endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose ?3′ and 5′ phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides.

The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

NMR structures of a nonapeptide from DNA binding domain of human polymerase-alpha determined by iterative complete-relaxation-matrix approach.

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-alpha, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by alpha, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (-3.1 x 10(-3)) of the fourth residue, 3J(NHalpha) coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, phi and psi dihedral angles for the i + 1, and i + 2 residues of the turn are found to be -80 and -41 and -60 and -40 degrees. The first proline residue is trans- while the second exists in both cis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5alpha-P6alpha correlation and phi and psi angles of the proline. The five-membered proline ring is in DOWN puckered (C-beta-exo/C-gamma-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are approximately 5 A(o) apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.     

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.