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

Abstract

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 sodium dodecyl sulfate (SDS) micelles. The final family of calculated structures displays three well-defined β- or γ-turn regions, comprising residues 1−5, 7−10, and 24−27, but has otherwise a random conformation. The receptor-interacting N-terminal part, residues 1−5, was found to be best defined with a backbone RMSD value of 0.12 Å. The mode of association between galanin and the SDS micelle was determined by observing the broadening effect on proton resonances, when spin-labeled 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 and 21, reaching out into the aqueous solvent. Additional NMR studies were carried out on an inactive analogue, Ala²−galanin, and an antagonist M40. The results show that the proton resonances of galanin and M40 have identical chemical shifts in the N-terminal receptor-interacting region, indicating similar solution structures in this region. For Ala²−galanin, the same region displays a spectral heterogeneity with chemical shifts clearly different from the other two peptides, indicative of different secondary structures. These results may provide a structural background for the antagonist activity of M40 and the hormonal inactivity of Ala²−galanin, as compared to galanin.