Nuclear-magnetic-resonance study on Met-enkephalin and Met-enkephalinamide. Molecular association and conformation

The 270-MHz 1H and 68-MHz 13C nuclear magnetic resonance spectra of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) and Met-enkephalinamide are analyzed in a variety of solvents. For the dipolar form of Met-enkephalin in (C2H3)2SO solution, significant concentration dependences are found of C-alpha proton chemical shifts, indicating an aromatic ring-current effect in molecular aggregates. An anomalous temperature dependence is observed of the amide proton chemical shift of the Met-5 residue. Furthermore, the chemical shifts of C-alpha protons of the dipolar form are found to depend appreciably on temperature. From the analyses of the temperature dependences together with concentration dependences of C-alpha proton resonances, the dipolar form of Met-enkephalin is found to be in an equilibrium of folded and extended conformations at low concentration in (C2H3)2SO solution. Solvent-composition dependences of the amide and C-alpha proton resonances and carbonyl and alpha-carbon resonances of the dipolar form in 2H2O/(C2H3)2SO solution are consistent with the conformation equilibrium and the association equilibrium. The folded conformation of the dipolar form in (C2H3)2SO solution is stabilized by the intramolecular attraction between the positively charged N-terminal group and negatively charged C-terminal group. The presence of the folded conformation is confirmed by the measurements of Gd(III)-induced relaxation enhancements of C-alpha protons. Nuclear Overhauser effects on the dipolar form are not consistent with the predominant formation of the beta-turn structure with the intramolecular hydrogen bond (Gly-2) C=O . H-N(Met-5). For the dipolar form of Met-enkephalin in 2H2O solution and for the cationic form of Met-enkephalinamide in (C2H3)2SO solution and in 2H2O solution there is no evidence for the formation of folded conformations.