Conformational analysis of CCK-B agonists using 1H-NMR and restrained molecular dynamics: comparison of biologically active Boc-Trp-(N-Me) Nle-Asp-Phe-NH2 and inactive Boc-Trp-(N-Me)Phe-Asp-Phe-NH2.
The tetrapeptide Boc-Trp-(N-Me)Nle-Asp-Phe-NH2 is a potent CCK-B agonist. Replacement in this analogue of the norleucine residue by a phenylalanine, to yield Boc-Trp-(N-Me) Phe-Asp-Phe-NH2, led to a 740-fold decrease in affinity whereas the same decrease in affinity was not observed in their nonmethylated counterparts. In order to ascertain the conformational preferences of these two N-methylated tetrapeptides, a study by two-dimensional (2D) nmr spectroscopy and molecular modeling was undertaken. The solution conformation of the two peptides was examined by 1H-nmr in a d6-DMSO/H2O (80:20) mixture. A cis-trans equilibrium, induced by N-methylation, was observed for both analogues, and the proton spectra of the two rotamers were fully characterized in each case. 1H-1H distance constraints, derived from 2D nuclear Overhauser effect spectroscopy and rotating frame nuclear Overhauser effect spectroscopy experiments, were used as inputs for subsequent restrained molecular dynamics simulations. Comparisons of the nmr and molecular modeling data point toward distinct conformational preferences for these two peptides with an opposite spatial orientation of the Trp residue, and could explain the large difference in their biological activities. Furthermore, the tridimensional structure of Boc-Trp-(N-Me)Nle-Asp-Phe-NH2 could serve as a model for the design of nonpeptide CCK-B agonists.
Goudreau
N
N
Laboratoire de Pharmacochimie Moléculaire et Structurale, U.266 INSERM, URA D1500 CNRS, Faculté de Pharmacie, Paris.
Weng
J H
JH
Roques
B P
BP
D000595:Amino Acid Sequence; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D011487:Protein Conformation; D011949:Receptors, Cholecystokinin / Q000037:antagonists & inhibitors*; D013816:Thermodynamics
D009842:Oligopeptides; D011949:Receptors, Cholecystokinin; C086503:t-butyloxycarbonyl-tryptophyl-(N-methyl)norleucyl-aspartyl-phenylalaninamide; C086502:tert-butyloxycarbonyl-tryptophyl-(N-methyl)phenylalanyl-aspartyl-phenylalaninamide