Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by H-1 NMR spectroscopy and molecular modeling calculation

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu -receptor lig and, in the biological function, the solution conformations of endomorphin- 2 and its C-terminal free acid (EM2OH, FI-Tyr-Pro-Phe-Phe-OH), studied usin g two-dimensional H-1 NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximate to 1:2. The lack of significant temperature and concentration dependence o f NH protons suggested that the NMR spectra reflected the conformational fe atures of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing me thod under the proton-proton distance constraints derived from the ROE cros s-peaks. These energy-minimized conformers, which were all in the phi torsi on angles estimated from J(NHC alphaH) coupling constants within +/- 30, we re then classified in groups one or two according to the folding backbone s tructures. All trans and cis EM2 conformers adopt an open conformation in w hich their extended backbone structures are twisted at the Pro(2)-Phe(3) mo iety. In contrast, the trans and cis conformers of EM2OH show conformationa l variation between the 'bow'-shaped extended and folded backbone structure s, although the cis conformers of its zwitterionic form are refined into th e folded structure of the close disposition of C- and N-terminal groups. Th ese results indicate clearly that the substitution of carboxyl group for C- terminal amide group makes the peptide flexible. The conformational require ment for p-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.