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

Abstract

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 ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H 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 approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.

Structural Investigation of Endomorphins by Experimental and Theoretical Methods: Hunting for the Bioactive Conformation

“…In contrast, this conformational feature of cis-EM1 was not observed during the MD calculation performed in H 2 O and in vacuo [25]. Similarly as for the cis-isomer of EM1, based on the NMR data, a compact sandwich structure with stacking of the Tyr 1 , Pro 2 , and Phe 3 rings was also detected for cis-EM2 in DMSO and in TFE [27] [29]. Moreover, the results of the RS calculations indicated the presence of compact conformations created by the packing of the side chains of aromatic amino acids around the Pro 2 residue in the case of the cis-isomers of EMs (Fig.…”

“…These results indicated that the replacement of the C-terminal amide group by a carboxy group resulted in a higher flexibility for EM2OH. The subsequent structural examination of EM2 and its C-terminal free-acid analog carried out in different media (i.e., H 2 O, DMSO, TFE, and DPC micelles) by In et al [27] led to the similar observations regarding the backbone structure of the cisand trans-isomers of both tetrapeptides, as described in [29]. In this latter study, four different types of backbone conformation were distinguished, namely the rS-and n7-type open conformers, as well as the F1-and F2-type folded conformers, whose population ratios depended on the solvent type and pH.…”

“…For EM2 and EM2OH, In et al [29] performed NMR measurements, followed by SA and MD simulations. On the basis of their results, it was concluded that all conformers of the cis-and trans-isomers of EM2 assumed a similar extended conformation with a twisted backbone at the Pro 2 -Phe 3 region (Fig.…”

“…Nevertheless, almost exclusively the trans-isomer of EM2 was observed in DPC micelles (pH 3.5 and 5.2) [27]. In the case of C-terminal free-acid analog (EM2OH), the NMR studies led to population ratios of cis-and trans-isomers which proved to be equivalent to those of EM2 in H 2 O (pH 2.7), DMSO, and DPC micelles (pH 3.5) [27] [29], but different proportions of the two isomers were detected in H 2 O (pH 5.2), TFE, and DPC micelles (pH 5.2) compared to those found for the parent peptide [27].…”

C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization

“…This may be related to the fact that amidation brings the C-terminal group to a neutral state, which in turn decreases the repulsion of carboxyl groups in the aspartic acid. This is known to play a key role in preventing the formation of a folded conformation for the C-terminal amidated peptide (46). An open conformation for OSN would allow more side chain interaction with calcium and phosphate ions (35).…”