Comparison of the solution structures of the chimeric peptides galanin(1–12)‐Ala‐neuropeptide Y(25–36)amide and galanin(1–12)‐Pro‐neuropeptide Y(25–36)amide

Abstract

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.

NMR Study of the Conformation and Localization of Porcine Galanin in SDS Micelles. Comparison with an Inactive Analog and a Galanin Receptor Antagonist

“…Such a γ-turn involves three residues, where the amide proton of the third residue is hydrogen-bonded to the carboxyl group of the first residue. In perfect agreement, the amide proton exchange rate is slow for Tyr 26 (Figure 3a). This structural arrangement brings the aromatic sidechains of both Phe 22 and Tyr 26 into the vicinity of Lys 25 and can explain the observed upfield shifted resonances of Lys 25 .…”

“…These regions coincide rather well with the structured parts found in the structure calculation, implying that these conformations may be relevant for recognition of the peptide by its CNS receptors. It should be mentioned that the two N-terminal parts of rat and porcine galanin are identical, while the C-terminal part differs (KHGLT [25][26][27][28][29] and KYGLA [25][26][27][28][29] for rat and porcine, respectively).…”

“…NMR spectroscopy has also been used to determine the solution structures of two galanin chimeras, galanin(1-12)-Ala-neuropeptide Y(25-36)-amide and galanin(1-12)-Proneuropeptide Y(25-36)-amide, in an aqueous solvent containing 30% 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) (25,26). The earlier paper described the N-terminal 12 residues, which are identical to the galanin sequence, as rather flexible with a reverse turn or a nascent helix located around Gly 8 and Tyr 9 .…”