High-Field NMR and Circular Dichroism Solvent-Dependent Conformational Studies of the Bradykinin C-Terminal Tetrapeptide Ser-Pro-Phe-Arg

Abstract

The conformational properties of the tetrapeptide Ser¹-Pro²-Phe³-Arg⁴, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H₂O and CD₃OH/D₂O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD₃OH/D₂O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe³ and Arg⁴ exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg⁴ and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH₃ ⁺(Ser¹)-COO^?(Arg⁴) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.