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 sodium dodecyl sulfate (SDS) micelles. The final family of calculated structures displays three well-defined beta- or gamma-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 A. 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.
Ohman
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A
Department of Biophysics, Stockholm University, Sweden.
Lycksell
P O
PO
Juréus
A
A
Langel
U
U
Bartfai
T
T
Gräslund
A
A
D000409:Alanine / Q000737:chemistry; D000577:Amides; D000595:Amino Acid Sequence; D000818:Animals; D019004:Galanin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D011522:Protons; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate; D013113:Spin Labels; D013552:Swine
D000577:Amides; C079517:M40; D008823:Micelles; D010446:Peptide Fragments; D011522:Protons; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide; D013113:Spin Labels; D012967:Sodium Dodecyl Sulfate; D019004:Galanin; D000409:Alanine