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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. Schwyzer (Biopolymers, 1995, Vol. 37, pp. 5-16). In both solvent systems, NPAF was found to be primarily alpha-helical within the central portion of the molecule, from Asn(6) to Ala(14). The N-terminus was random in both solvent systems. In the SDS solution, the C-terminal tetrapeptide was structured and formed a type I beta-turn, whereas in TFE/H(2)O it was unstructured, showing the importance of the C-terminal tetrapeptide in receptor recognition. NPAF was found to associate with SDS, and was shown to be near the surface of the micelle by spin label studies with 5-doxyl-stearic acid., (Copyright 2003 Wiley Periodicals, Inc.)

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. F5C, pentafluorocinnamoyl, is the N-terminal protecting group and is not involved in the peptide secondary structure. B-10324 contains an N-terminal Pro(2)- CpG(5) distorted type II beta-turn whereas the rest of the peptide is random. A salt bridge is not observed between the carboxylate group at the C-terminal end and the Arg(1) side chain, in contrast to that previously observed for B-9858 and B- 10148. The conformations are correlated with the measured B(1) receptor antagonist activities (J.-F. Larrivée, L. Gera, S. Houle, J. Bouthillier, D. R. Bachvarov, J. M. Stewart and F. Marc au, Br. J. Pharmacol. 131, 885-892 (2000)). The importance of the N-terminal beta-turn is highlighted.

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.

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 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.