A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.