MIMICKING THE MEMBRANE-MEDIATED CONFORMATION OF DYNORPHIN A-(1-13)-PEPTIDE - CIRCULAR-DICHROISM AND NUCLEAR-MAGNETIC-RESONANCE STUDIES IN METHANOLIC SOLUTION

The structural requirements for the binding of dynorphin to the kappa-opioid receptor are of profound clinical interest in the search for a powerful nonaddictive analgesic. These requirements are though to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13)-peptide, Tyr1-Gly2-Gly3-Phe4-Leu5-Arg6-Arg7-Ile8-Arg9-Pro10-Lys11-Leu12-Lys13. Schwyzer has proposed an essentially alpha-helical membrane-mediated conformation of the 13 amino acid peptide [Schwyzer, R. (1986) Biochemistry 25, 4281-4286]. In the present study, circular dichroism (CD) studies on dynorphin A-(1-13)-peptide bound to an anionic phospholipid signified negligible helical content of the peptide. CD studies also demonstrated that the aqueous-membraneous interphase may be mimicked by methanol. The 500- and 620-MHz H-1 nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13)-peptide in methanolic solution were sequence-sspecifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY (DQF-COSY), relayed COSY (RELAY), and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg7 to Arg9 was in extended or beta-strand conformation, which agreed with deuterium-exchange and temperature-dependence studies of the amide protons and analysis of the vicinal spin-spin coupling constants 3J(HN-alpha). The results clearly demonstrated the absence of extensive alpha-helix formation. Chi-1 rotamer analysis of the 3J-alpha-beta demonstrated no preferred side-chain conformations.