NMR-STUDY OF THE CONFORMATION AND LOCALIZATION OF PORCINE GALANIN IN SDS MICELLES - COMPARISON WITH AN INACTIVE ANALOG AND A GALANIN RECEPTOR ANTAGONIST

Galanin is a 29/30-residue neuro-endocrine peptide which performs its many important physiological functions via a membrane-bound receptor. By using two-dimensional proton NMR spectroscopy, complete relaxation matrix analysis, and simulated annealing, the conformation of porcine galanin was determined in a membrane-mimicking solvent containing sodi um dodecyl sulfate (SDS) micelles. The final family of calculated stru ctures displays three well-defined beta- or gamma-turn regions, compri sing residues 1-5, 7-10, and 24-27, but has otherwise a random conform ation. The receptor-interacting N-terminal part, residues 1-5, was fou nd to be best defined with a backbone RMSD value of 0.12 Angstrom. The mode of association between galanin and the SDS micelle was determine d by observing the broadening effect on proton resonances, when spin-l abeled 5- and 12-doxyl stearate molecules were added. It was concluded that galanin is located close to the surface of the micelle with two regions, residues 6-9 and 24-29, as well as two single residues, 18 an d 21, reaching out into the aqueous solvent. Additional NMR studies we re carried out on an inactive analogue, Ala(2)-galanin, and an antagon ist M40. The results show that the proton resonances of galanin and M4 0 have identical chemical shifts in the N-terminal receptor-interactin g region, indicating similar solution structures in this region. For A la2-galanin, the same region displays a spectral heterogeneity with ch emical shifts clearly different from the other two peptides, indicativ e of different secondary structures. These results may provide a struc tural background for the antagonist activity of M40 and the hormonal i nactivity of Ala2-galanin, as compared to galanin.