A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYINLITRQRY-NH2, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15 degrees C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined alpha-helix in the centre, with a few residues at the termini having less well defined conformations. The spin-lattice and spin-spin relaxation rates of alpha-carbons have been determined on 13C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C alpha-H alpha vectors in the alpha-helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.

A hybrid peptide of cecropin A and melittin was investigated by two-dimensional 'H-NMR at pH 5.8 in aqueous solution with 30% (by vol.) hexafluoroisopropanol. The peptide contains 26 amino acids, is a combination of the first 13 residues of each of the two parent peptides, CA(1-13)M(1-13) = CAM (1-26) 1061 and shown to have strong antibacterial activity but to be harmless towards erythrocytes. All resonances of the main chain and side chain /I-protons are assigned except for those of the N-terminal lysine. Several medium range NOE connectivities were observed showing two separated a-helices, involving residues 4-12 and 16-26. The J,,,-coupling constants in these sections support the conclusion. From the exchange rates of the NH protons it is concluded that the a-helix of residues 16 -26 is much more stable than the other helix. The circular dichroism data indicates about 30% less a-helix character than the NMR data. A reduced contribution to the ellipticity from the unstable helix is suggested.The chemical-shift differences between the two parts of the hybrid and the respective parent peptides are larger for the cecropin part than for the melittin part. For the latter, residues 17-26 of the hybrid are proposed to have a secondary structure very similar to that of residues 4-13 of melittin. The total results suggest that a hydrophobic a-helix capable of spanning half a lipid bilayer combined with an amphiphilic a-helix of two to three turns with a flexible hinge section in between are features of importance for the lytic antibacterial activity.

The 25 amino acid residue chimeric peptide M32, galanin(1-13)-neuropeptide Y(25-36)-amide, was synthesized. The peptide was found to be recognized by both galanin and NPY receptors. The solution structure in 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol was examined by 2-D 1H-NMR and by CD. Proton resonance assignments were made, and structures were calculated using DIANA and refined by restrained energy minimization and molecular dynamics. The obtained structures contain an alpha-helical part in the NPY portion of the peptide including residues 13-20, and in some structures it continues to the C-terminal Tyr25. The more flexible N-terminal portion of the peptide has the freedom to approach the C-terminal alpha-helix, via a reverse turn or a nascent alpha-helix, which permits the N-terminus with Trp2 to come into close contact with the C-terminus with Tyr25. Among the ten NMR structures with lowest energy, there are structures reminiscent of the horseshoe shape of aPP, a close relative of NPY with known crystal structure. It appears that the strong alpha-helical character of the NPY (25-36) amide fragment of M32 helps to stabilize structural features in the galanin-derived part of the peptide. It is noteworthy that this rigid NPY portion of M32 does not prevent the recognition of the peptide by galanin receptors; rather, the peptide has unusually high affinity: IC50 = 0.1 nM at galanin receptors. The chimeric peptide M32 is also recognized by NPY receptors with submicromolar affinity (IC50 = 0.25 microM). The availability of a solution structure for peptide M32, which is recognized by two peptide receptors that are both members of the family of G-protein-coupled receptors, may be useful in understanding peptide receptor-ligand interactions and in designing new galanin and NPY receptor ligands.

The solution structure of the vasoactive endogenous 21-amino-acid human endothelin-2 has been determined by NMR and CD techniques, in a mixed solvent of 100 mM aqueous acetic acid and 25% (by vol.) 1,1,1,3,3,3-hexafluoro-2-propanol. From NMR-derived restraints on upper limit distances and dihedral angles, distance geometry structures were calculated using the program DADAS90, and refined by simulated annealing in X-PLOR. The structure of endothelin-2 consists of an A-helix of residues 9 to 17, orientated anti-parallel to a short β-strand of residues 1 to 3, linked together by a possible β-turn type I of residues 5Ϫ8. These secondary structural elements are stabilised and positioned by two disulphide bonds between residues 1 and 15, and 3 and 11, respectively. The average root mean square deviation over residues 1Ϫ17 of 15 accepted low-energy conformers chosen to reflect the solution structure of endothelin-2, was 0.73 Å for the backbone and 1.41 Å for all heavy atoms. The data on endothelin-2 will be discussed and compared with what has been published on other endothelin/sarafotoxin peptides.Keywords : endothelin-2; NMR; circular dichroism ; solution structure ; helix.Endothelin-1 was first isolated from porcine aortic endothelial cells [1]. The human genes encoding this peptide and two isoforms, endothelin-2 and endothelin-3, have been encountered and cloned [2]. From analysis of a mouse genome another peptide called vasoactive intestinal contractor (VIC) was identified [3]. These four mammalian peptides are strongly homologous ( Fig. 1). Moreover, they have pronounced sequence similarities with the snake venom sarafotoxins [4] and Bibrotoxin [5]. For this reason they have been suggested to form an endothelin/sarafotoxin peptide family. These peptides have 21 residues and two disulphide bonds connecting residues 1 and 15, and 3 and 11, respectively. They are also noted for evoking similar biological actions [6].The endothelin peptides were first characterised as being the most potent constrictors of smooth muscle. They are now known to produce a variety of pharmacological effects in various vascular and non-vascular tissues and to show numerous activities in the central and peripheral nervous systems. They have been linked to regulation of blood flow and to a number of diseases such as vasospasm, hypertension, myocardial infarction, arteriosclerosis and renal failure [7]. The role as a vasoconstrictor has however been questioned after a finding that blood pressure of transgenic 'knock-out' mice with only one copy of the gene, is somewhat raised, rather than lowered as was expected [8]. Disruption of both copies of the gene results in an embryonic lethal phenotype with malformed craniofacial organs [8].Two G-protein-coupled endothelin receptors have been characterised and cloned in mammalian species, ET A and ETB [9,Correspondence to H. Nakanishi, Biomolecular Chemistry Laboratory, Department of Biomolecules, National Institute of Bioscience and Human-Technology (NIBH), 1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8...

Biochemistry 32, 7787 -77981, the 25-amino-acid-residue chimeric peptide M32, galanin( 1 -12)-Pro-neuropeptide Y(25-36)amide, was found to bind very strongly to galanin receptors and also to have considerable affinity to neuropeptide Y receptors. The solution structure of M32 in 30% (by vol.) 1,1,1,3,3,3-hexafluoro-2-propanol, was determined from structural restraints obtained by twodimensional 'H-NMR. Another peptide, M88, with Ala instead of Pro in position 13, was shown to bind with tenfold lower affinity to galanin receptors, i.e. with nearly the same affinity as galanin itself. The binding to neuropeptide Y receptors was altered in the opposite sense. The peptide M88 has now been examined by two-dimensional 'H-NMR under the same conditions as applied for M32. Using NMR-derived restraints, we have calculated structures of M88 by means of the programs CALIBA, HABAS and DIANA, and refined them by restrained energy minimisation and restrained molecular dynamics. The use of CALIBA and HABAS meant that the restraints were less restrictively formulated, than in the previous work. Hence, structures of M32 were recalculated in the same way as for M88, also including some additional restraints, possible to identify by comparison of the NMR spectra of the two peptides. The new structures of M32 confirm an a-helix starting at Prol3, but now continuing until Gln23, instead of ending at Ile20. Conversely, no secondary structure is now expressed in the N-terminal section. It is concluded that in the case of peptides the details of the calculated structures depend on how the restraints are calibrated. In M88, an a-helix is found over a long section, approximately Ser6 -Gln23. Possible correlations between the binding affinities to receptors and the solution structures of the peptides M32 and M88 are discussed. This is done with particular reference to the length and stability of the a-helix and the flexibility of the N-terminal section and its bending direction versus the helix.It has been found that peptides composed of biologically active fragments of different neuropeptides may show interesting properties different from those observed for the fragments themselves. In particular, in receptor binding studies some chimeric peptides have revealed much enhanced affinities for the natural receptors (Gozes et al., 1991 ;Bartfai et al., 1991; Arvidsson et al., 1993), and in biological tests IC,,, concentration of competitive inhibitor that inhibits SO% of the binding of a radioligand to a receptor; DQF, double quantum filtered ; DANTE, delays alternating with nutations for tailored excitation; COSY, correlated spectroscopy; ECOSY, exclusive COSY; CALIBA, calibration of NOE intensity versus distance constraints ; HABAS, program for obtaining stereo-specific resonance assignments for a-and p-protons in proteins; DIANA, distance geometry algorithm for NMR applications ; REDAC, redundant dihedral angle constraints ; rEM, restrained energy minimisation ; rMD, restrained molecular dynamics.