A PROTON MAGNETIC-RESONANCE STUDY OF 2 SYNTHETIC AGONIST ANTAGONIST PAIRS OF BRADYKININ ANALOGS

The conformation of two agonist-antagonist pairs of bradykinin (Arg1-P ro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) analogues were studied in CD3O H/H2O solution by H-1-nmr techniques. The first agonist peptide studie d, Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-Pro7-Thi8-Arg9, differs from the bradykinin sequence by the addition of D-Arg0, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = beta-(2-thienyl)-L-al anine), and Hyp3 (Hyp = L-4-hydroxy-L-proline) in position 3. In the c orresponding antagonist sequence, Pro7 is replaced by D-Phe7. The seco nd agonist-antagonist pair studied does not contain the D-Arg0 residue , which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two-dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresid ue, sequential, and medium-range nuclear Overhauser effects, amide pro ton temperature coefficients, and vicinal coupling constants. Both ago nist peptides show clear evidence for the existence of a type I beta-t urn comprising the C-terminal residues Ser6-Pro7-Thi8-Arg9 in fast con formational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the pres ence of the beta-turn is spectroscopically clearly discernible. The tw o antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with so me irregularities in the N-terminal region of the peptide. While the e xistence of a turn at the C-terminal end of bradykinin and its analogu es with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first ti me, provides evidence based on physical data in a polar solvent enviro nment that the turn is present, that it is type I and that it is essen tial for agonist activity. In the particular solvent used in these stu dies, the Pro7 to D-Phe7 substitution precluded the formation of the t urn for the C-terminal residues of the antagonist.