Ion binding of cyclolinopeptide A: an NMR and CD conformational study

CD and nmr techniques have been used to study, in acetonitrile solution, the ion-complexing capability of cyclolinopeptide A (CLA), a cyclic nonapeptide of sequence cyclo-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val) endowed with remarkable cytoprotective ability in vitro, and the conformation of the Ba(2+)/CLA complex. At room temperature, CLA in acetonitrile shows a proton nmr spectrum characteristic of the coexistence of many different conformers in intermediate exchange. The backbone contains a cis Pro-Pro bond, with all other peptide bonds in the trans conformation. CLA binds Ba2+ more tightly than the other cations studied, namely K+, Na+, Mg2+, and Ca2+; CD data are indicative of the presence of both 1:2 (sandwich) and 1:1 (equimolar) type complexes, depending on the Ba2+ ion concentration, whereas nmr data are consistent with an equimolar form. The relevant conformational features of the equimolar Ba2+/CLA complex are that the backbone contains all trans peptide bonds, a type I 6----3 beta-turn and a 3----1 gamma-turn (or a distorted 3----9 beta-turn). The global shape of the complexed peptide can be described as a bowl, with the concave (polar) side hosting Ba2+ and the convex side predominantly apolar.     

Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z)Δ]3-Gly4)], as seen by two-dimensional 1H-nmr spectroscopy

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L -MeAla¹-L -Leu²-MePhe[(Z)Δ]³-Gly⁴ )] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at ?5°C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180° rotation of a nonmethylated peptide bond. © 1995 John Wiley & Sons, Inc.     

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.     

Conformations of blood group H-active oligosaccharides of ovarian cyst mucins

Spectroscopic data and conformational energy calculations are reported for eight oligosaccharides from ovarian cyst mucins and from human milk, the nonreducing terminals of which have fucose (α1 → 2)galactose linked either (β1 → 3) (type I) or (β1 → 4)(type II) to N-acetylglucosamine or in (β1 → 3) linkage to galactosaminitol. The fully assigned proton nmr spectra are reported along with nuclear Overhauser enhancement (NOE) data. Amide proton coupling constants and vacuum-uv CD spectra provide information on the amide plane orientation and amide environment. Our results imply that the fucosidic dihedral angles are similar for all three cases and that the substantial differences in the chemical shifts of the fucosyl protons of type I, type II, and 3-ol chains result from different perturbations by the amide group of the residue to which the β-galactose is linked. Stereopair diagrams of conformational models for both type I and II H chains are presented that are consistent with NOE, coupling constants, conformational energy calculations, and the CD data. While the temperature dependence of the observed NOE of penta- and hexasaccharides indicates that their rotational correlation times are strongly temperature dependent, we conclude that the conformations are essentially independent of temperature.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

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.     

Advantages of fine-grained side chain conformer libraries

We compare the modelling accuracy of two common rotamer libraries, the Dunbrack-Cohen and the 'Penultimate' rotamer libraries, with that of a novel library of discrete side chain conformations extracted from the Protein Data Bank. These side chain conformer libraries are extracted automatically from high-quality protein structures using stringent filters and maintain crystallographic bond lengths and angles. This contrasts with traditional rotamer libraries defined in terms of chi angles under the assumption of idealized covalent geometry. We demonstrate that side chain modelling onto native and near-native main chain conformations is significantly more successful with the conformer libraries than with the rotamer libraries when solely considering excluded-volume interactions. The rotamer libraries are inadequate to model side chains without atomic clashes on over 20% of targets if the backbone is held fixed in the native conformation. An algorithm is described for simultaneously modelling both main chain and side chain atoms during discrete ab initio sampling. The resulting models have equivalent root mean square deviations from the experimentally determined protein loops as models from backbone-only ensembles, indicating that all-atom modelling does not detract from the accuracy of conformational sampling.     

A conformational study of the xyloglucan oligomer, XXXG, by NMR spectroscopy and molecular modeling

A structural study of the XXXG xyloglucan heptasaccharide (X = alpha-D-Xylp(1 --> 6)-beta-D-Glcp and G = beta-D-Glcp) isolated from apple fruit has been undertaken with nmr and molecular mechanics methods. Quantitative 400 MHz nmr data including nuclear Overhauser effect spectroscopy (NOESY) volumes were recorded at both 6 and 20 degrees C. In spite of severe overlapping of resonances, it was possible to estimate summed NOEs for the majority of the anomeric and glucosyl methylene protons. An ensemble-average population of preferred geometries has been established with the CICADA conformational searching algorithm associated with the MM3 force field. Comparison of the theoretical data obtained by back-calculation of the NOESY volumes from the ensemble-average distance matrix program and motional models based on the Stokes-Einstein-Debye relation satisfactorily reproduce the experimental data. Conformational averaging about the mainchain glycosidic linkages includes both the syn and anti conformers and a minor gauche-gauche population is highly probable. The theoretical data overestimate the syn preference of the Glc(c) --> Glc(b) linkage as well as the Glc(c) GT rotamer population. Finally, both the motional models and the conformational search indicate a fairly rigid backbone and greater flexiblity for the xylose side chains.     

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.     

Fluorescence, CD, and NMR studies on spantide, a bombesin and substance P antagonist.

The conformational properties of spantide [(D-Arg, D-Trp, Leu) substance P] have been studied by fluorescence, CD, and nmr techniques. The fluorescence, CD, and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for bombesin. A preliminary investigation on [D-Pro] spantide is also reported.     

Conformational preferences of Leu-enkephalin in reverse micelles as membrane-mimicking environment

Enkephalin represents one of the bioactive peptide molecules most extensively investigated both in solution and in the crystal state. Depending upon the environment chosen for such studies, three main conformational states were identified for this flexible, linear pentapeptide—i.e., an extended conformation, a single-bend, and a double-bend structure. Since CD and Fourier transform ir (FTIR) spectra of Leu-enkephalin solubilized in negatively charged reverse micelles of bis (2-ethylhexyl)sulfosuccinate sodium salt/isooctane/water were supportive of a restricted conformational space of the aromatic side chains and of a bended type fold, we have analyzed by nmr the conformational preferences of Leu-enkephalin in reverse micelles using a synthetic [¹³C, ¹⁵N]-backbone-labeled sample. The overall conformation derived from nuclear Overhauser effect spectroscopy (NOESY) and ¹⁵N-filtered rotating frame NOESY (ROESY) spectra and by distance geometry calculations is a double-bend fold of the backbone that is comparable to one of the known x-ray structures. Thereby the tyrosine side chain is inserted into the hydrophobic core of the reverse micelles in a restrained conformational space as well evidenced by NOEs between the aromatic ring protons and the surfactant. The proximity of the aromatic rings of tyrosine and phenylalanine indicate a preferred structure consistent with the postulated conformation of the opioid peptide in the δ-receptor-bound state. These results confirm the interesting and promising properties of reverse micelles as membrane mimetica. © 1997 John Wiley & Sons, Inc. Biopoly 41: 591–606, 1997     

Synthesis and conformational studies by 1H- and 13C-NMR spectroscopy of a novel, sterically constrained analogue of thyrotropin-releasing hormone

A novel thyrotropin-releasing hormone (TRH) analogue, [2,4-MePro3]-TRH (2,4-MePro: 2-carboxy-2,4-methanopyrrolidine), has been synthesized using a rapid solid phase peptide synthesis method, and its conformational properties investigated by one- and two-dimensional (2D) nmr spectroscopy and by proton Overhauser measurements. Following a published approach, calibrated interproton Overhauser effects were used together with distance geometry analysis to deduce that the single conformation of the His-2,4-MePro tertiary amide bond is trans in aqueous solution. This conclusion was corroborated by 2D dipolar-correlated (NOESY) spectroscopy. A preferentially extended conformation is indicated by the nmr data, similar to that of TRH. The phi, psi conformational space of 2,4-MePro is, however, significantly different from that of trans proline and the structural consequences of these differences at the C-terminus are discussed. The distribution of histidine side-chain conformations in the TRH analogue was deduced from coupling constants and from the short-range interaction between the imidazole ring and one of the prochiral faces of the 2,4-MePro side chain.     

Modifications of the amide bond and conformational constraints in pseudopeptide analogues

We have investigated the conformational effects of modifying the amide group in model dipeptides. The N-methyl amide ψ[CO-NMe], N-hydroxy amide ψ[CO-N(OH)], N-amino amide ψ[ CO-N (NH₂)], retro amide ψ[ NH-CO], reduced amide in the neutral ψ[CH₂-NH] and protonated ψ[CH₂-N ⁺ H₂] state, and hydrazide ψ[CO-NH-NH] have been introduced as surrogates of the amide link in pseudopeptide derivatives of the Pro-Gly or Ala-Gly model dipeptides protected on both termini by an amide group. These compounds have been studied in solution by proton nmr and ir spectroscopy, and in the solid state by x-ray diffraction, giving an extended data set of experimental structural and conformational information on pseudopeptide sequences. The conformational effects depend both on the nature and the position of the modified amide link. Some modifications appear to have no intrinsic conformational induction (N-amino and retro amide), but destabilize any local folded structure by hydrogen-bond breaking. Because of the formation of strong intramolecular interactions, others are capable of stabilizing a β-turn (for example protonated reduced amide), or of inducing a particular local conformation such as a β- or γ-like turn (for example N-hydroxy amide). The particular geometry of the cis N-methyl amide and of the “hydrazino” proline favors the formation of a sharp turn of the main chain. All these structural data are of interest to the design of bioactive peptide mimics. © 1993 John Wiley & Sons, Inc.     

Conformational flexibilities in malformin A

The linked-atom-least-squares (LALS) technique has been applied to generate exactly cyclized and stereochemically satisfactory conformations of the cyclic pentapeptide, malformin A, which contains an intramolecular disulfide bridge across a D-Cys—D-Cys linkage. Consistent with theoretical, ir, and x-ray evidence from studies on analogs of the LKiysteinyl—L-cysteine disulfide, it is shown that the peptide bond across the S? S bridge in malformin A can retain a cis configuration. The two all-trans structures proposed earlier by A.E. Tonelli [(1978) Biopolymers 17, 1175–1179] from solution nmr data have also been analyzed. Both P-(χ^s ～ 90°) and M-(χ^s ～ ?90°)-type helicity of the S? S bridge are found to be accessible in both the trans and the cis models in which the respective conformations of the homodetic rings are essentially preserved. The details of six different conformational states and their relative energies have been evaluated and compared. Our findings, which suggest that a variety of conformational states are accessible to malformin A, are compatible with other published results from solution studies. On the basis of hydrogen-bonding interactions, a model is proposed to explain how malformin A might be inactivated specifically by L -Hyp, and not by L -Pro or other amino acids, as has been observed from in vivo investigations [Buckhout, T.J. & Curtis, R.W. (1976) Nature 260 , 435–436]     

Contrasting solution conformations of peptides containing α,α-dialkylated residues with linear and cyclic side chains

The conformational properties of α,α-dialkylated amino acid residues possessing acyclic (diethylglycine, Deg: di-n-propylglycine, Dpg; di-n-butylglycine, Dbg) and cyclic (1-amino-cycloalkane-1-carboxylic acid, Ac_nc) side chains have been compared in solution. The five peptides studied by nmr and CD spectroscopy are Boc-Ala-Xxx-Ala-OMe, where Xxx = Deg(I). Dpg (II), Dbg (III), Ac₆c (IV), and Ac₇c (V). Delineation of solvent-shielded NH groups have been achieved by solvent and temperature dependence of NH chemical shifts in CDCl₃ and (CD₃)₂SO and by paramagnetic radical induced line broadening in pepiide III. In the Dxg peptides the order of solvent exposure of NH groups is Ala(1) > Ala(3) > Dxg(2), whereas in the Ac_nc peptides the order of solvent exposure of NH groups is Ala(1) > Ac_nc(2) > Ala(3). The nmr results suggest that Ac_nc peptides adopt folded β-turn conformations with Ala(1) and Ac_nc(2) occupying i + 1 and i + 2 positions. In contrast, the Dxg peptides favor extended C₅ conformations. The conformational differences in the two series are clearly borne out in CD studies. The solution conformations of peptides I-III are distinctly different from the β-turn structure observed in crystals. Low temperature nmr spectra recorded immediately after dissolution of crystals of peptide II provide evidence for a structural transition. Introduction of an additional hydrogen-bonding function in Boc-Ala-Dpg-Ala-NHMe (VI) results in a stabilization of a consecutive β-turn or incipient 3₁₀-helix in solution. © 1995 John Wiley & Sons, Inc.     

Conformations of cyclic peptide/calcium complexes in solution

Synthetic cyclic octapeptides of general structure cyclo[Glu(γOBzl)-Sar-Gly-(N-R)Gly]₂ (R = n-hexyl and cyclohexyl) transport calcium ions selectively across organic phases and phospholipid membranes. We have now used proton nmr spectroscopy (360 MHz) to study the solution conformation(s) of their calcium complexes. When Ca(ClO₄)₂ was added to solutions of these peptides in CDCl₃, nmr spectra of the resulting calcium complexes were characteristic of a single C₂-symmetric conformer. From a Karplus-Bystrov analysis of vicinal coupling constants in both the peptide backbone and Glu side chain (treated as an ABCC′MX spin system), in conjuction with model-building studies, a structure was proposed in which the calcium ion is bound in an octahedral-type complex by the four (coplanar) carbonyl groups of the (all-trans) Glu-Sar and Gly-(N-R)Gly peptide bonds. Occurrence of preferred rotamers about Glu side chain C^α–C^β bonds indicated that restricted rotation in peptide side chains arises upon calcium binding.     

Conformational studies of a short linear peptide corresponding to a major conserved neutralizing epitope of human respiratory syncytial virus fusion glycoprotein

The conformational properties of a 21-residue peptide, corresponding to amino acids 255 to 275 (F255-275) of the human respiratory syncytial virus fusion (F) glycoprotein, have been studied by CD and nmr spectroscopy. This peptide includes residues 262, 268, and 272 of the F polypeptide that are essential for integrity of most epitopes that mapped into a major antigenic site of the F molecule. CD data indicate that F255-275 adopts a random coil conformation in aqueous solution at low peptide concentrations. However, as the concentration of peptide is increased, a higher percentage of peptide molecules adopts an organized structure. This effect can be more easily observed when trifluoroethanol (30%) is added to peptide solutions, giving rise to CD spectra that resemble those of α-helix structures. These conformational changes were confirmed by nmr spectroscopy. The nuclear Overhauser effects observed in 30% trifluoroethanol/water together with the conformational H_α chemical shift data allowed us to propose a structural model of helix-loop-helix for the peptide in solution. In addition, these helical regions contain the amino acid residues essential for epitope integrity in the native F molecule. These results give new insights into the antigenic structure of the respiratory syncytial virus F glycoprotein. © 1996 John Wiley & Sons, Inc.     

Chemical properties of the histidine residue of secretin: evidence for a specific intramolecular interaction

Secretin has a single histidine residue located at the amino terminus which plays a crucial role in its biological activity. The chemical properties, viz. pK and reactivity, of the alpha-amino and imidazole groups of this residue were determined at a secretin concentration of 10(-6) M in 0.1 M KCl at 37 degrees C. Competitive labelling using tritiated 1-fluoro-2,4-dinitrobenzene (DNP-F) as the labelling reagent was the experimental approach employed. The alpha-amino group was found to have a pK value of 8.83 and a reactivity 5-times that of the alpha-amino group in the model compound, histidylglycine. For the imidazole function a pK value of 8.24 and a reactivity 26-times that of the imidazole function in histidylglycine was found. Both these groups in secretin had pK values which were shifted one pK unit higher than in histidylglycine, but like the model compound the reactivity of the imidazole function was still linked to the state of ionization of the alpha-amino group. These observations are interpreted as evidence for the existence of a major conformational state in dilute aqueous solution in which the amino-terminal histidine of secretion is interacting with a negatively charged carboxyl group.     

The solution conformation of malformin A

Solution conformations of the cyclic pentapeptide plant-hormone malformin A, whose conformational freedom is constrained by an intramolecular disulfide bridge, are derived and presented here. The nmr and CD data of Ptak are used to place restrictions on the search for possible malformin A solution conformers of low energy. Only two distinct conformers were found to be consistent with Ptak's data. Both structures are characterized by an internally buried (solvent-shielded) D -Cys² amide proton, a seven-membered (1–3)hydrogen bond between (N–H) and (O?C), and a disulfide bridge conformation with a P chirality as manifested in the nmr study by the temperature independence of the amide proton chemical shifts for the D -Cys² and D -Leu⁴ residues and the negative sign of the long wavelength maximum in the CD spectrum, respectively. Inspection of space-filling molecular models of both structures indicates severe steric barriers to their rapid interconversion. Thus, it appears that only one of the two conformers may be present in solution. The difference in their calculated dipole moments (4.6 and 6.9D) suggests an experimental method for distinguishing between the two proposed solution structures.     

Helix formation in model peptides based on nucleolin TPAKK motifs

The structures formed by peptide models of the N-terminal domain of the nucleolar protein nucleolin were studied by CD and nmr. The sequences of the peptides are based on the putative nucleic acid binding sequence motif TPAKK: The peptides TP1 and TP2 have the sequence acetyl-G(ATPAKKAA)_nG-amide, with n = 1 and 2, respectively. CD measurements indicate structural changes in both peptides when the lysine side chains are uncharged by increasing the pH or acetylation of the side-chain amines. When trifluoroethanol (TFE) is added, more extensive structural changes are observed, resembling helical structure based on nmr nuclear Overhauser effect (NOE) and C^α proton chemical shift changes, and CD spectra. The structure formed in 0.5M NaClO₄ as observed by nmr is similar to that when the lysine side chains are acetylated, due presumably to interactions of perchlorate ion with side-chain charges on lysines. The helical structure observed in TPAKK motifs may be stabilized via N-capping interactions involving threonine. The structures observed in TFE suggest that the Thr-Pro sequence initiates short helical segments in TPAKK motifs, and these helical structures might interact with nucleic acids, presumably via interactions between lysines and threonines of nucleolin. © 1995 John Wiley & Sons, Inc.