A peptide corresponding to a portion of the amphipathic alpha-helical region of CTP:phosphocholine cytidylyltransferase was synthesized. This region of the enzyme was proposed to be the membrane-binding domain [Kalmar, G. B., Kay, R. J., Lachance, A., Aebersold, R., & Cornell, R. B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6029]. We have shown that the peptide is physically associated with PG vesicles. CD of the peptide in buffer suggested a primarily random structure, while, in the presence of trifluoroethanol, the peptide was alpha-helical. Anionic lipid vesicles promoted an alpha-helical conformation, where as neutral or cationic lipid vesicles did not alter the random structure of the peptide, suggesting a selective stabilization of the alpha-helix by anionic membranes. The fluorescence of the single tryptophan residue, which lies on the hydrophobic face of the amphipathic alpha-helix, was studied. Anionic lipid vesicles specifically induced a shift in the fluorescence to a lower wavelength. Fluorescence quenching by the aqueous-phase quencher, I-, and the lipid-phase quencher 9,10-dibromo-PC was used to determine the accessibility of the tryptophan to each of these environments. The presence of anionic lipid vesicles, but not nonanionic lipid vesicles, decreased the quenching by I- suggesting that, in the presence of anionic lipids, the tryptophan residue is poorly accessible to the aqueous I-. Dibromo-PC significantly quenched the fluorescence only when present in anionic vesicles, confirming the membrane location of the tryptophan residue and the lipid specificity of this interaction. 9,10-Dibromo-PC quenched the fluorescence more efficiently than did 6,7-dibromo-PC or 11,12-dibromo-PC, localizing the tryptophan to the same depth in the bilayer as the ninth carbon on a fatty acid chain. These studies show that this peptide binds selectively to anionic membranes in an alpha-helical conformation, and the binding involves intercalation of the hydrophobic face of the helix into the membrane core. Thus, we propose that the mechanism of cytidylyltransferase activation by membranes involves lipid-specific stabilization of an amphipathic helical structure in the C-terminal region of the protein.
