The Membrane-Mediated Conformation of Dynorphin A-(1-13)-Peptide as Studied by Nuclear Magnetic Resonance Spectroscopy, Circular Dichroism Spectropolarimetry, and Molecular Dynamics

Abstract

The structural requirements for the binding of dynorphin to the kappa opioid receptor are of profound clinical interest in the search for a powerful non-addictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13}, Tyr¹-Gly²-Gly³-Phe⁴-Leu⁵-Arg⁶-Arg⁷-Ile⁸-Arg⁹-Pro¹⁰-Lys¹¹-Leu¹²-Lys¹³. Schwyzer [𝘉𝘪𝘰𝘤𝘩𝘦𝘮𝘪𝘴𝘵𝘳𝘺 25: 4281-4286 (1986)] has proposed an essentially α-helical membrane-mediated conformation of the tridecapeptide. In the present study, the hydrophobic moment, the helix probability and a four -state secondary structure prediction were computed. They signified, in agreement with circular dichroism (CD) studies on phospholipid-bound dynorphin A-(1-13)-tridecapeptide, negligible helical content of the peptide. CD studies demonstrated that the aqueous-membraneous interphase can be mimicked by methanol. The 500 and 620 MHz ¹H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13) in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive 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 an extended or β-strand conformation, which was in line with deuterium exchange and temperature dependence studies of the amide protons. ¹³C_α spin-lattice relaxation rate constants indicated a non-rigid backbone conformation. Transferred nuclear Overhauser effect studies on aqueous systems containing dynorphin A-(1-13) in the presence of dimyristoyl-phosphatidylcholine bilayers indicated a folded conformation from Tyr¹ to Leu⁵. The findings were incorporated into a tentative molecular model, which also indicated a non-helical, non-extended conformation for the rest of the molecule in the presence of corresponding distance-restrained negative charges.