Backbone Structure Confirmation and Side Chain Conformation Refinement of a Bradykinin Mimic BKM-824 by Comparing Calculated 1H, 13C and 19F Chemical Shifts with Experiment

Abstract

Calculated and experimental 1H, 13C and 19F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava1-Igl2-Ser3-DF5F4-Oic5-Arg6] (Ava=5-amino- valeric acid, Igl=α-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)- octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947–955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6–31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6–311G** basis set. There is an intramolecular hydrogen bond at Ser3 in the optimized structures. The experimental 13C chemical shifts at five Cα positions as well as at the Cβ, Cγ and Cδ position of Ava1, which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental Hβ chemical shifts in Igl2 shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the χ1 angle in DF5F4 suggest that χ1 ≈180°. The calculated 1H and 13C chemical shifts are in good agreement with experiment at the rigid residue Oic5. None of the models gives accurate results for Arg6, presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.