title: 1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.
abstract: A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale U91, Hôpital Henri Mondor, Créteil, France.
authors.forename: B
authors.initials: B
authors.lastname: Prêcheur
authors.forename: H
authors.initials: H
authors.lastname: Munier
authors.forename: J
authors.initials: J
authors.lastname: Mispelter
authors.forename: O
authors.initials: O
authors.lastname: Bârzu
authors.forename: C T
authors.initials: CT
authors.lastname: Craescu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D013552:Swine
chemical_list: D002147:Calmodulin; D009587:Nitrogen Isotopes; D010455:Peptides; D006859:Hydrogen