Mimicking the membrane-mediated conformation of dynorphin A-(1-13)-peptide: circular dichroism and nuclear magnetic resonance studies in methanolic solution

• Published in: Biochemistry (Easton) Vol. 30; no. 19; pp. 4715 - 4726
• Main Authors: Lancaster, C. Roy D, Mishra, Prasanna K, Hughes, Donald W, St.-Pierre, Serge A, Bothner-By, Aksel A, Epand, Richard M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 14-05-1991
• Subjects: 550201 - Biochemistry- Tracer Techniques; Aminoacids, peptides. Hormones. Neuropeptides; ANALGESICS; Analytical, structural and metabolic biochemistry; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; Cell Membrane; CENTRAL NERVOUS SYSTEM DEPRESSANTS; Circular Dichroism; CONFORMATIONAL CHANGES; DICHROISM; DRUGS; Dynorphins - chemistry; Dynorphins - metabolism; Fundamental and applied biological sciences. Psychology; MAGNETIC CIRCULAR DICHROISM; MAGNETIC RESONANCE; Magnetic Resonance Spectroscopy; MEMBRANE PROTEINS; Methanol; MOLECULAR STRUCTURE; N.M.R; NUCLEAR MAGNETIC RESONANCE; ORGANIC COMPOUNDS; phospholipids; Protein Conformation; PROTEIN ENGINEERING; PROTEINS; Receptors, Opioid - metabolism; Receptors, Opioid, kappa; RESONANCE; Solutions; STRUCTURE-ACTIVITY RELATIONSHIPS; TEMPERATURE DEPENDENCE; Ligand binding; Circular dichroism spectrometry; COSY sequence; Overhauser effect; Dynorphin A; NMR spectrometry; Opioid peptide; Conformation; Methanol; Biological receptor
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00233a012

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 thought 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 1H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13)-peptide in methanolic solution were sequence-specifically 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 an 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 3JHN 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.