STRUCTURAL AND BIOCHEMICAL-STUDIES OF HUMAN GALANIN - NMR EVIDENCE FOR NASCENT HELICAL STRUCTURES IN AQUEOUS-SOLUTION

The 30-residue human neuropeptide, galanin, was shown to bind to rat i nsulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insuli n secretion from these cells in a manner quantitatively similar to tha t of porcine, galanin. Neither human nor porcine galanin stimulated Ca 2+ mobilization in cultured human small cell lung carcinoma cells. Sed imentation equilibrium analysis of human galanin showed that it was st rictly monomeric in aqueous solution, indicating that the peptide inte racts with its receptor(s) as a monomer. The monomeric nature of the p eptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on g alanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equili brium with random coil. At 33 degrees C, the short-range structures in clude a nascent helix spanning residues 3-11 which incorporates a hydr ophobic core from residues 6-11. Residues 14-18 and 22-30 display sequ ential NH-NH and (CH)-H-beta-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the ct-region of conformational space. However, no medium-r ange dipolar connectivities indicative of nascent helix or turn confor mations were observed. At 3 degrees C, almost all residues significant ly populate the cr-region of conformational space, and the nascent hel ix between residues 3 and 11, with its hydrophobic core, is retained. As expected, circular dichroism (CD) was insensitive to the presence o f short-range structure, and therefore the CD spectrum of human galani n in aqueous solution indicated a completely random coil peptide. Howe ver, changes in the CD spectrum resulting from the addition of 30% (v/ v) of the helix-promoting organic solvent, trifluoroenthanol, indicate d that similar to 6 residues of the peptide were transformed to stable helix.