A CD-STUDY 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 se quence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, wit h sequence D-Arg-Arg-Pro-Hyp-Gly-Thi-D-Ser-D-Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L-proline; Thi refers to beta-(2-thienyl)-L-alanine and Cpg refers to alpha-cyclopentylglycine) . The effect of increasing concen tration of TFE in water on the difference ellipticity at 222 nm was ex amined and showed that BK may be a mixture of at least two different c onformers, one of which largely forms when the TFE concentration is in creased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreeme nt with that shown by the BK antagonist, indicating that the conformat ion of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both on e-dimensional and two-dimensional H-1-nmr techniques. The total correl ation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H2O so lution at 10 degrees C and a nuclear Overhauser effect spectroscopy (N OESY) spectrum that shows only sequential H-alpha(i) - NH (i + 1) or t he H-alpha(i) - H-delta delta'(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY f or BK in the 95/5% (v/v) TFE/H2O solution shows that there are two maj or conformations in the solution with about equal population. The NOES Y experiment shows two new important cross peaks for one conformation, namely Pro(2)(alpha)-Pro(3)(alpha) and the Pro(2)(alpha)-Gly(4)(NH), indicating a cis Pro(2)-Pro(3) bond and a type VI beta-turn between re sidues Arg(1) and Gly(4) involving cis proline at position 3, respecti vely. The low temperature coefficient of Gly(4) for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa beta-turn is present. The other conformation is all trans and extended. The BK antagonist shows difference CD spectra in TFE soluti ons referred to H2O that are superficially indicative of a beta-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-t rans extended conformation over the range of TFE concentrations. The B K-antagonist CD data suggest that solvent perturbation of the CD of an extended conformation mimics the formation of a hydrogen-bonded struc ture. This is thought to arise from the perturbation of the optical ac tivity of the thienyl moiety of the peptide since the CD spectrum of N -acetyl-beta-thienyl-L-alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relations hip between CD and nmr. (C) 1994 John Wiley & Sons, Inc.