NMR conformational analysis of proadrenomedullin N-terminal 20 peptide, a proangiogenic factor involved in tumor growth.

The preferred conformation of Proadrenomedullin N-Terminal 20 Peptide (PAMP; ARLDVASEFRKKWNKWALSR-amide) has been determined using 1H and 13C two-dimensional nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. PAMP is a peptide that has various physiological functions, including its role as a proangiogenic factor in facilitating tumor growth and its inhibitory effect on catecholamine secretion at nicotinic receptors. The preferred conformation of PAMP was determined in a helix-inducing trifluoroethanol and water (TFE/H2O) solution, and in a membrane-mimetic sodium dodecylsulfate-d25 (SDS) micellar solution. The secondary structure consists of an alpha-helix for residues Arg2 to Arg20 in TFE/H2O solution and an alpha-helix for residues Arg2 to Ala17 in SDS solution. We postulate that the polar charged residues Arg2, Lys12, and Arg20 are responsible for the initial interaction of the peptide with the micelle, and that this is followed by the binding of the hydrophobic residues Leu3, Val5, Phe9, Trp13, and Trp16 to the micellar core. The three C-terminal amino acid residues adopt an extended structure in SDS, suggesting that they are important in receptor recognition and binding. This is supported by truncation studies done by Mahata et al. (Hypertension, 1998, Vol. 32, pp. 907-916), which show the importance of the C-terminal in physiological activity. Furthermore, Belloni et al. (Hypertension, 1999, Vol. 33, pp. 1185-1189), and Martinez et al. (Cancer Research, 2004, Vol. 64, pp. 6489-6494) suggested that the N-terminal was also important in PAMP activity. However, no differences in conformational preference of the N-terminal were observed between the two solvent systems.

The importance of the N-terminal beta-turn in bradykinin antagonists.

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.

An NMR conformational analysis of cyclic bradykinin mimics. Evidence for a beta-turn.

A detailed NMR study is carried out in acetonitrile/water solutions on three novel cyclic bradykinin antagonist analogues, BKM-824, BKM-870, and BKM-872, to examine their solution structures, and to correlate the structures with bradykinin antagonist and anti-cancer activities. The solution structures of the cyclic peptides are correlated with the structural data for known linear bradykinin antagonists. The sequences are: BKM-824 c[Ava-Ig1-Ser-DF5F-Oic-Arg] where Ava is 5-aminovaleric acid, Ig1 is alpha-(2-indanyl)glycine, F5F is pentafluorophenylalanine, and Oic is (2S,3aS,7aS)-octahydroindole-2-carboxylic acid; BKM-870; c[DArg-Arg-Add-DF5F-Oic-Arg] where Add is 12-aminododecanoic acid; and BKM-872; c[DArg-Arg-Eac-Ser-DF5F-Oic-Arg] where Eac is 6-aminocaproic acid. BKM-824 was the only peptide within this series that possessed a discernable solution structure. The NMR data indicate the presence of a type I beta-turn between residues F5F4 and Ava1, a C-terminal-like end. Molecular dynamics calculations show that a type I beta-turn from DF5F4 to Ava1 does exist although the turn was somewhat distorted. This result differs from the structures seen in linear bradykinin antagonists, which usually possess a type II'beta-turn at the C-terminal end and the presence of a defined turn is correlated with bradykinin antagonist activity. There is no solution structure for BKM-870 and BKM-872 but a correlation between the primary sequence Arg(terminal)-DArg1-Arg2-long chain aliphatic amino acid and anti-cancer activity is evident.

An nmr conformational analysis of a synthetic peptide Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from the New World Centruroides noxius 2 (Cn2) scorpion toxin: comparison of the structure with those of the Centruroides scorpion toxins.

The solution structure of a synthetic peptide, Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from toxin 2 (Cn2) of the New World scorpion Centruroides noxius was determined using nmr and molecular dynamics calculations. The peptide has no significant secondary structure such as an alpha-helix or a beta-sheet, yet it has a fixed conformation for the first chain. The backbone secondary structure involving residues 6-12 in this peptide shows an excellent overlap with the structures of natural neurotoxins from Centruroides sculpturatus Ewing. Residues 6-9 form a distorted type I beta-turn and residues 10-12 form a gamma-turn. As residues 7-10 in the Centruroides toxins correspond to one of the regions of highest sequence variability, it may account for the species specificity and/or selectivity of toxic action. The conformation of this region evidently plays an important role in receptor recognition and in binding to the neutralizing monoclonal antibody BCF2 raised against the intact toxin.

Information from the water stripe in TOCSY experiments on systems with exchangeable protons.

The water "stripes" of the TOCSY maps of aqueous solutions of sucrose, of a 15-aminoacid peptide, and of several of the constituent aminoacids are shown to contain correlations at the resonance frequencies of protons which are scalar coupled to OH or NH protons which exchange with the solvent. Theoretical analysis of chemical exchange during the spin-lock period in TOCSY elucidates the origin of these correlations, and shows that their intensities vary with the duration of the spin-lock period and with the exchange rate.

NMR and CD conformational studies of bradykinin and its agonists and antagonists: application to receptor binding.

Most physiological processes are regulated by peptides that perform their functions by interacting with specific receptors on cells. Specific conformations of the peptides are required for correct interactions to take place, and a knowledge of the biologically important conformation is vital for the understanding of biological function. Over the last few years extensive studies using nuclear magnetic resonance and circular dichroism have been carried out on bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and its antagonists with the objective of developing new drugs to combat severe pathologies associated with its production. In the present review, these techniques for the determination of peptide conformation are reviewed and applied to the study of bradykinin and its antagonists. Modeling of these conformational data in the presence of the B2 receptor or an antibody allows the biologically active conformations to be deduced and these are presented in this review.

A comparative NMR and molecular dynamics study of the conformations of bradykinin B1 and B2, B2, and B1-specific receptor antagonists B-9430, B-9436, and B-9858.

Extensive proton magnetic resonance experiments were carried out on three bradykinin peptide antagonists B-9430, B-9436, and B-9858 in aqueous solutions as well as in sodium dodecylsulphate micelles (B-9430 and B-9436) and CD3OH/H2O (60%/40%) mixtures for B-9858. All three peptides showed no observable secondary structure in aqueous solution. However, in their respective structure-inducing solvents, B-9430 (B1 and B2 receptor antagonist) and B-9436 (a B2 receptor antagonist) exhibit a type II beta-turn involving residues 2-5, and B-9430 also exhibits a type II' beta-turn involving residues 6-9 (in sodium dodecylsulfate micellar solutions), whereas B-9858, a B1-specific receptor antagonist, exhibits only a type II beta-turn involving residues 2-5 (in CD3OH/H2O solutions). Simulated annealing calculations on B-9858 confirm the experimental conclusions based on the nmr data. In addition, simulated annealing of the (2S, 3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic residue), which is present in two of the three decapeptides studied, show that the one-chair conformation of the six-membered ring predominates, in agreement with the experimental data. The activities of these peptides are compared with their secondary structures and the specific receptor activity appears to depend on the presence of specific amino acid residues, such as N-(2-indanyl) glycine (Nig) and D[alpha-(2-indanyl) glycine] (D-Igl) as well as on elements of secondary structure.

An NMR, CD, molecular dynamics, and fluorometric study of the conformation of the bradykinin antagonist B-9340 in water and in aqueous micellar solutions.

A detailed NMR, CD, fluorometry, and molecular modeling study of a novel bradykinin antagonist B-9340, containing a novel amino acid D-Igl (alpha-(2-indanyl)glycine) at position 7, was carried out. The sequence of B-9340 is D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D- Igl7-Oic8-Arg9, where Hyp is hydroxyproline, Thi is beta-(2-thienyl)alanine, and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid. The CD results exhibit a striking effect of SDS on the spectrum of the BK antagonist, indicating that interaction with the surfactant induces a folded peptide structure. The interaction of this antagonist with phosphatidylinositol was monitored by fluorometry, indicating that the interaction of the peptide with the lipid is cooperative, and gives a Hill coefficient of 2.3. The two-dimensional proton NMR measurements indicate that B-9340 has no stable secondary structure in water solution and contains about 10-15% cis peptide bonds arising from Pro2, Hyp3, and Oic8. In SDS micelles, NMR reveals the existence of two beta-turns based on a number of medium-range connectivities that were useful for molecular modeling. The actual molecular modeling and dynamic runs were performed on B-9340 in an environment consisting of a layer of octyl sulfate anions and water. Ther results indicate that the structure of B-9340 in a micellar environment is characterized by a nonideal betaII-turn comprising residues Pro2 to Thi5, a nonideal betaII'-turn comprising residues Ser6-Arg9, and broad folding in the middle part of the molecule. The structure is stabilized by several hydrogen bonds and by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9, whereas the middle part of the peptide is buried in the micelle. The structure is deposited as Brookhaven PDB file 1 BDK.

Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides.

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type 1 beta turn involving the same amino acids in both glycosylated and unglycosylated peptides. The alpha GalNAc attached to the threonine (T3) and serine (S4) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.

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 beta-(2-thienyl)-L-alanine and Cpg refers to alpha-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 degrees C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H alpha (i)-NH(i + 1) or the H alpha (i)-H delta delta' (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 (alpha)-Pro3 (alpha) and the Pro2 (alpha)-Gly4(NH), indicating a cis Pro2-Pro3 bond and a type VI beta-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 beta-turn is present. The other conformation is all trans and extended. The BK antagonist shows difference CD spectra in TFE solutions referred to H2O that are superficially indicative of a beta-bend.(ABSTRACT TRUNCATED AT 400 WORDS)

Conformational analysis of the type II and type III collagen alpha-1 chain C-telopeptides by 1H NMR and circular dichroism spectroscopy.

The type II and type III collagen alpha-1 chain C-telopeptides are a 27 mer with the sequence NAc-GPGIDMSAFAGLGPREKGPDPLQYMRA and a 22mer,NAc-GGGVASLGAGEKGPVGYGYEYR, respectively. Their conformations have been studied in CD3OH/H2O (80/20) solution by means of two-dimensional proton NMR and CD spectroscopy. Based on TOCSY and NOESY experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H alpha coupling constants, sequential and medium range NOEs and amide proton temperature coefficients. The conformation of the type II C-telopeptide is essentially extended. Evidence from CD spectroscopy suggests that a very minor proportion of the peptide might be helical (ca.8%), but the NMR data show no evidence for a non-linear structure. The observation of reduced amide proton temperature dependence coefficients in certain sections of the molecule can, in view of the absence of any other supporting evidence, only be interpreted in terms of local shielding from solvent for sterical reasons (large hydrophobic side-chains). The conformation of the type III C-telopeptide is mostly extended except for a beta-turn ranging from Gly8 to Glu11, which is stabilized by a hydrogen-bond between NH of Glu11 and the carbonyl group of Gly8. The low temperature coefficient of NH(Glu11) and, in particular, the observation of a medium range NOE between H alpha (A9) and NH(E11) corroborate the existence of a beta-turn in this region. Although spectral overlap prevents a precise conclusion with regard to the type of beta-turn present, there is some evidence that it might be type II.

Conformational analysis of the type II and type III collagen alpha-1 chain N-telopeptides by 1H-NMR spectroscopy and restrained molecular mechanics calculations.

The type II and type III collagen alpha-1 chain N-telopeptides are a nonadecamer with the sequence pEMAGGFDEKAGGAQLGVMQ-NH2 and a tetradecamer with the sequence pEYEAYDVKSGVAGG-NH2, respectively. Their conformations have been studied in CD3OH/H2O (60/40) solution by means of two-dimensional proton nmr spectroscopy. Based on double quantum filtered correlation spectroscopy, total correlation spectroscopy, rotating frame nuclear Overhauser enhancement (ROE) spectroscopy, and nuclear Overhauser enhancement (NOE) spectroscopy experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H alpha coupling constants, sequential and medium-range NOEs (ROEs), and amide proton temperature coefficients. The NOE distance constraints as well as dihedral constraints based on the vicinal NH-H alpha coupling constants were used as input parameters for restrained molecular mechanics, consisting of restrained molecular dynamics and restrained energy minimization calculations. The type II N-telopeptide's conformation is dominated by a fused beta gamma-turn between Phe6 and Ala10, stabilized by three hydrogen bonds and a salt bridge between the side-chain end groups of Glu8 and Lys9. The first 5 amino acids are extended with a much higher degree of conformational freedom. The 2 Gly residues following the turns were found to be highly flexible (hinge-like), leaving the spatial position of the second half of the molecule relative to the fused beta gamma-turn undefined. In the type III telopeptide, a series of sequential NH(i)-NH(i + 1) ROEs were observed between the amino acids Tyr2 and Ser9, indicating that a fraction of the conformational space is helical. However, the absence of medium-range ROEs and the lack of regularity of the effects associated with alpha-helices suggest the presence of a nascent rather than a complete helix.

Proton magnetic resonance studies of bradykinin antagonists.

Bradykinin (BK) is a peptide hormone with sequence Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9 and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, beta-branched D-aliphatic residues at position 7 combined with bulky L-aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D-Arg0,Hyp3,Thi5,D-Cpg7,Cpg8]-BK [Cpg: alpha-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L-proline; Thi: beta-(2-thienyl)-L-alanine] (I), [D-Arg0,Hyp3,D-Cpg7,Cpg8]-BK (II), as well as its variant with D-Cpg7 replaced by Cpg7, namely [D-Arg0,Hyp3,Cpg7,Cpg8]-BK (III). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II, in direct correlation with the peptide activities. No turn-like structure was found for residues 6-9. In peptide III, a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists (I, II) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D-Cpg7]-BK (IV) showed no defined secondary structure.

The aggregation properties of some bradykinin analogs.

Bradykinin (BK) is a peptide hormone with sequence Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9 and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. Bulky, beta-branched D-aliphatic residues at position 7 combined with bulky L-aliphatic residues at position 8 have now been observed to yield strong antagonists. Nuclear magnetic resonance studies have been carried out on many of these molecules with a view to determining their solution conformations. However, two such analogs, namely DArg-[Hyp3, Thi5, DSer6, DCpg7, Cpg8]-BK [I] and DArg-[Hyp3, DSer6, DCpg7, Cpg8]-BK [II] (Cpg = alpha-cyclopentyl-glycine; Hyp = 4-hydroxy-L-proline, Thi = beta-(2-thienyl)-L-alanine), have exhibited an abnormal, non-linear temperature dependence for the amide NH proton of Cpg8. The NH of Arg9 also shows a slightly non-linear temperature dependence at higher temperatures above 25 degrees C. In addition, a very slow exchange rate for the NH protons of DCpg7, Cpg8 and Arg9 indicated aggregation of these two analogs, which was confirmed using the circular dichroism experiments.

A proton magnetic resonance study of two synthetic agonist-antagonist pairs of bradykinin analogues.

The conformation of two agonist-antagonist pairs of bradykinin (Arg1-Pro2-Pro2-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) analogues were studied in CD3OH/H2O solution by 1H-nmr techniques. The first agonist peptide studied, D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-Pro7- Thi8-Arg9, differs from the bradykinin sequence by the addition of D-Arg0, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = beta-(2-thienyl)-L-alanine), and Hyp3 (Hyp = L-4-hydroxy-L-proline) in position 3. In the corresponding antagonist sequence, Pro7 is replaced by D-Phe7. The second agonist-antagonist pair studied does not contain the D-Arg0 residue, which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two-dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresidue, sequential, and medium-range nuclear Overhauser effects, amide proton temperature coefficients, and vicinal coupling constants. Both agonist peptides show clear evidence for the existence of a type I beta-turn comprising the C-terminal residues Ser6-Pro7-Thi8-Arg9 in fast conformational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the presence of the beta-turn is spectroscopically clearly discernible. The two antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with some irregularities in the N-terminal region of the peptide. While the existence of a turn at the C-terminal end of bradykinin and its analogues with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first time, provides evidence based on physical data in a polar solvent environment that the turn is present, that it is type I and that it is essential for agonist activity. In the particular solvent used in these studies, the Pro7 to D-Phe7 substitution precluded the formation of the turn for the C-terminal residues of the antagonist.

A sequence-dependent 1H-NMR study on the formation of beta-turns in tetrapeptides containing charged residues.

The importance of side-chain charge interactions in the formation of beta-turns was studied. Sixteen protected NAc-tetrapeptide amides were studied, namely the variants of DEKS: NEKS, EEKS, DDKS, DQKS, NQKS, DERS, NERS, EERS, DDRS, NDRS, DQRS, and DKES. Three tetrapeptides--NPDM, NSDM, and NDDS--were also studied as they have a high probability of forming beta-turns, based on statistical predictions. The results indicate that a small proportion of type I beta-turn exists in solutions of DEKS and DERS in methanol/water (60/40), while NEKS has an even smaller population of this turn. The other tetrapeptides are present in solution only in the extended conformation. These results clearly show the importance of the salt bridge between the side chains of K2 and E3 or R2 and E3, as well as the importance of the charge on the side chain of the first residue in stabilizing the beta-turn. The relevance of statistical predictions for beta-turns in short peptides is discussed.

The solution conformation of tubulin-beta(422-434)-NH2 and its Nac-DATADEQG-NH2 fragment based on NMR.

The solution conformation of tubulin-beta(422-434)-NH2 (YQQYQDATADEQG-NH2) and its Nac-DATADEQG-NH2 fragment has been studied by two-dimensional 1H-nmr spectroscopy in CD3OH/H2O (90/10 v/v) at neutral and low pH. The 13 amino acid peptide is a segment of the C-terminal region of tubulin, and is directly involved in the selective binding site with microtubule-associated proteins MAP-2 and the tau protein. Based on correlated spectroscopy, total correlation spectroscopy, and rotating frame nuclear Overhauser effect spectroscopy experiments, a complete assignment of all proton resonances was achieved, and the conformation of the backbone could be deduced from coupling constants, NH temperature coefficients, and nuclear Overhauser effects. The spectroscopic evidence indicates that the T8-Q12 section of both molecules forms one complete alpha-helical turn, stabilized by a NH (Q12)-C = O (T8) hydrogen bond. Furthermore, strong pH-dependent backfolding of the E11 side chain to its own NH proton was found. In addition, close proximity between the aromatic side chains of Y1, Y4, and the alpha-helical part, resulting in some substantial chemical shift changes when comparing the entire 13-mer with the octamer, could be explained in terms of a nonclassical kink in the DATA section. The conformational space is dominated by extended structures and the nonextended conformers are only a minor, yet spectroscopically clearly discernible entity. The presence of the alpha-helical region at the C-terminus of the 13-mer is important because binding studies of this peptide with MAP-2 indicate that the D10-E11-Q12-G13 fragment is critical for the binding interaction.

Solution conformation of the type I collagen alpha-2 chain telopeptides studied by 1H and 13C NMR spectroscopy.

The high-field 1H and 13C NMR studies of the N- and C-terminal telopeptides of the alpha-2 chain of collagen were carried out in CD3OH/H2O solutions. All proton assignments are based on two-dimensional phase-sensitive COSY and ROESY experiments. The conformation of the N-telopeptide (nonamer) is predominantly extended with a small proportion of the molecules existing in a type I beta turn. The four residues involved in this turn are D3-A4-K5-G6 which is stabilized by a C = O(D3)-NH(G6) hydrogen bond. The C-terminal telopeptide is extended throughout. A model is proposed involving charge-charge and hydrophobic interactions between the extended alpha-2 chain N-telopeptide and the adjacent segments of triple-helix. A similar model is proposed for the C-telopeptide.

A 1H and 13C NMR study on the role of salt-bridges in the formation of a type I beta-turn in N-acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2.

The conformation of the tetrapeptide N-Acetyl-Asp7-Glu8-Lys9-Ser10-NH2, a fragment of the type I collagen alpha-1 chain N-telopeptide, has been studied by 1H and 13C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD3OH/H2O (60/40) at ca. neutral pH the tetrapeptide forms a beta-turn, stabilized by a hydrogen bond between NH(S10) and CO(D7) and a strong salt-bridge between COO-(E8) and NH3+(K9). The beta-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H alpha (E8) coupling constant, NH(E8) low-field shift and the temperature coefficient of NH(S10), whereas the conclusion regarding the salt-bridge is based on 13C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E8-K9 charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.