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 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.
Lancaster
C R
CR
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Mishra
P K
PK
Hughes
D W
DW
St-Pierre
S A
SA
Bothner-By
A A
AA
Epand
R M
RM
D002462:Cell Membrane; D002942:Circular Dichroism; D004399:Dynorphins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D000432:Methanol; D011487:Protein Conformation; D011957:Receptors, Opioid / Q000378:metabolism; D017473:Receptors, Opioid, kappa; D012996:Solutions
D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D012996:Solutions; D004399:Dynorphins; D000432:Methanol