1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

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.