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.

NMR conformational studies of micelle-bound orexin-B: a neuropeptide involved in the sleep/awake cycle and feeding regulation.

The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH(2)) has been determined by (1)H and (13)C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d(25) (SDS), was used to mimic a physiological environment for the peptide.

The NMR-derived conformation of orexin-A: an orphan G-protein coupled receptor agonist involved in appetite regulation and sleep.

The conformation of orexin-A, an orphan G-protein coupled receptor agonist has been determined when bound to sodium dodecylsulphate-d(25) (SDS) micelles by (1)H and (13)C NMR and molecular modeling. Orexin-A has been implicated in sleep-wakefulness and feeding regulation. The conformational preference of orexin-A consists of a short helical section, involving Asp(5) to Gln(9) that makes up helix I, followed by a bend from Lys(10) to Ser(13).

The NMR-derived conformation of neuropeptide AF, an orphan G-protein coupled receptor peptide.

The tertiary structure of the pain modulating and anti-opiate neuropeptide, human neuropeptide AF (NPAF) (the sequence is AGEGLNSQFWSLAAPQRF-NH(2)), was determined by (1)H-NMR. The structure of NPAF was determined in two solvent systems, namely 50%/50% trifluoroethanol-d(3)/H(2)O (TFE/H(2)O) and in the cell membrane mimetic micelle, sodium dodecylsulfate-d(25) (SDS). The receptor for NPAF is an orphan G-protein coupled receptor, and the micellar SDS solvent system was used to emulate the cell membrane surface in line with the Cell Membrane Compartments Theory proposed by R.

Backbone structure confirmation and side chain conformation refinement of a bradykinin mimic BKM-824 by comparing calculated (1)H, (13)C and (19)F chemical shifts with experiment.

Calculated and experimental (1)H, (13)C and (19)F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava(1)-Igl(2)-Ser(3)-DF5F(4)-Oic(5)-Arg(6)] (Ava=5-aminovaleric acid, Igl=alpha-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)-octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al.

The NMR-derived conformation of neuropeptide F from Moniezia expansa.

The solution structure of neuropeptide F (NPF), from the flatworm (platyhelminthes) Moniezia expansa, has been determined by (1)H NMR spectroscopy at 800 MHz in 60%/40% CD(3)OH/H(2)O. NPF is the most abundant neuropeptide in platyhelminthes. The secondary structure of NPF contains an alpha helix from residues Lys(14) to Ile(31), while the N terminus, consisting of residues Pro(-2) to Asn(13), and the C-terminus, consisting of residues Gly(32) to Phe(36), are in a random conformation.

Correlation of secondary structures of bradykinin B1 receptor antagonists with their activity.

The secondary structure of a bradykinin B(1)receptor antagonist B-10324 (F5C-Lys-(1)- Lys(0)-Arg(1)-Pro(2)- Hyp(3)-Gly(4)-CpG(5)- Ser(6)-DTic(7)-CpG(8)) was determined by NMR at 800MHz. The conformational data are compared with those obtained previously for two bradykinin B(1) receptor antagonists, namely B-9858 (Lys-(1)- Lys(0)-Arg(1)-Pro(2)- Hyp(3)-Gly(4)-Igl(5)- Ser(6)-DIgl(7)-Oic(8)) and B-10148 (Lys-(1)-Lys(0)-Arg(1)- Pro(2)-Hyp(3)-Gly(4)- Igl(5)-Ser(6)-DF5F(7)- Oic(8)). The abnormal amino acids are: Hyp, trans-4- hydroxyproline; Tic, 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid; Oic, (2S, 3aS, 7aS)-octahydroindole-2-carboxylic acid; Igl, alpha(2- indanyl)glycine; F5F, 2,3,4,5,6-pentafluorophenylalanine; CpG, alpha- cyclopentylglycine.

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.

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.

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.

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.

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.

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).

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.

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.

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%.

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.

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.

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.

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.

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.

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 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.