Threonine 6 -Bradykinin: Structural Characterization in the Presence of Micelles by Nuclear Magnetic Resonance and Distance Geometry † Maria Pellegrini, ‡,§ Stefano Mammi, § Evaristo Peggion, § and Dale F. Mierke* ,‡,| Gustaf H. Carlson School of Chemistry, Clark University, 950 Main Street, Worcester, Massachusetts 01610, Department of Organic Chemistry, University of Padova, Biopolymer Research Center, Via Marzolo 1, Padova, Italy I-35131, and Department of Pharmacology and Molecular Toxicology, University of Massachusetts, Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655 Received July 25, 1996 X The conformation of the natural peptide [Thr 6 ]-bradykinin, Arg 1 -Pro 2 -Pro 3 -Gly 4 -Phe 5 -Thr 6 -Pro 7 - Phe 8 -Arg 9 , is investigated by NMR spectroscopy and computer simulations in an aqueous solution of sodium dodecyl sulfate micelles. The structural analysis of the peptide is of particular interest since it displays a different biological profile from bradykinin despite the high sequence homology (only one conservative substitution: Ser 6 /Thr 6 ) and the fact that both peptides bind and activate common receptors. The SDS micelles provide a model system for the membrane- interface environment the peptide experiences when interacting with the membrane-embedded receptor and allow for the conformational examination of the peptide using high-resolution NMR techniques. The NMR spectra show that the micellar system induces a secondary structure in the otherwise inherently flexible peptide (as observed in benign aqueous solution). The distance geometry calculations indicate a -turn of type I about residues 7-8 as the preferred conformation. The results of ensemble calculations reveal conformational changes occurring rapidly on the NMR time scale and allow for the identification of three different families of conformations that average to reproduce the NMR observables. The three families differ in the type of conformation adopted at the C-terminus: type I -turn, type II -turn and a third conformation, intermediate between the two -turns. The structural results support the hypothesis of the determining role of the C-terminal conformation for biological activity and can provide an explanation of the different activities observed for bradykinin and [Thr 6 ]- bradykinin. Introduction Bradykinin (BK; Arg 1 -Pro 2 -Pro 3 -Gly 4 -Phe 5 -Ser 6 -Pro 7 - Phe 8 -Arg 9 ) is a linear nonapeptide hormone produced by enzymatic cleavage of its high molecular weight precursor, kininogen, at the occurrence of tissue injury or trauma. 1 The role of the peptide in the regulation of major physiological systems as well as in a wide variety of pathological responses has been demonstrated (for a recent review, see ref 2). It is one of the most potent vasodilators and increases vascular permeability. 3,4 BK also elicits contraction of smooth muscles of the respira- tory and gastrointestinal tract and the uterus. 5 BK is active in the central nervous system, where it initiates pain stimuli 6 and is responsible for the cardinal symp- toms of inflammation. 7 Recently BK has been associ- ated with the symptoms of the common cold. 8,9 The conformational analysis of BK, BK fragments, and analogs is the object of considerable interest with the aim of gaining insight into a possible bioactive conformation and development of a structure-activity relationship. The general conclusion of conformational studies in aqueous solution is that BK exists in many conformational states. 10 However, in alternative solvent systems the inherently flexible nonapeptide preferred folded conformations. In the absence of X-ray crystal- lography or NMR data on the structure of the peptide when complexed to its receptor, one must rely on such studies, possibly in a solvent that mimics the biological environment the peptide experiences when bound to the receptor, to establish possible bioactive secondary struc- ture(s). The receptor for BK (at least two classes have been identified) 2 belongs to the family of G-protein-coupled receptors, characterized by seven transmembrane hy- drophobic helical segments. A membrane-bound path- way for the interaction between the peptide hormones and their receptors has been hypothesized; 11,12 the mechanism implies the accumulation and orientation of the peptide on the membrane, thus increasing the local concentration and at the same time reducing the degrees of rotational and translational freedom (i.e., reduction from 3D diffusion to a lateral, 2D dif- fusion). 13-15 This could also facilitate the transition from the random coil structure, usually adopted by the peptides in the extracellular solution, to the bioactive conformation. 16-18 The NMR study of peptides incorporated into mem- branes is extremely difficult, owing to the drastic line broadening, the high concentration of the lipid compared to the embedded peptide, and the overlap of signals. Therefore micellar systems have been often used to mimic a membrane-like environment which may induce biologically relevant conformations in oligopeptides 19,20 and is suitable for NMR studies. 21-26 † Abbreviations: BK, bradykinin; [Thr 6 ]-BK, threonine 6 -bradykinin; NMR, nuclear magnetic resonance; NOEs, nuclear Overhauser en- hancements; SDS, sodium dodecyl sulfate; DG, distance geometry; DADD, distance- and angle-driven dynamics. * To whom correspondence should be addressed at Clark University. Tel: (508) 793-7220. Fax: (508) 793-8861. E-mail: dmierke@clarku.edu. ‡ Clark University. § University of Padova. | University of Massachusetts. X Abstract published in Advance ACS Abstracts, December 15, 1996. 92 J. Med. Chem. 1997, 40, 92-98 S0022-2623(96)00539-0 CCC: $14.00 © 1997 American Chemical Society