Ca2+-myristoyl switch and membrane binding of chemically acylated neurocalcins

Neurocalcin is a member of a novel family of neuronal calcium sensors that belongs to the superfamily of EF-hand Ca2+-binding proteins. Neurocalcin is myristoylated on its N-terminus in vivo and can associate with biological membranes in a calcium and myristoyl-dependent manner. This process known a s "Ca2+-myristoyl switch" has been best described for the photoreceptor spe cific protein, recoverin, as well as for several other neuronal calcium sen sors. Here, we used reversed micelles to chemically acylate nonmyristoylate d neurocalcin at its N-terminus with fatty acids of different lengths (from C12 to C16). This approach allowed us to prepare neurocalcin derivatives i n which a single fatty acid is selectively linked to the N-terminal glycine of the polypeptide chain through an amide bond. The membrane binding prope rties of the monoacylated neurocalcins were then examined by cosedimentatio n with phospholipid vesicles and direct binding to lipid monolayers by surf ace plasmon resonance spectroscopy (Biacore). Our results show that neuroca lcins monoacylated with lauric, myristic, or palmitic acid were able to ass ociate with membrane in a calcium-dependent manner. This indicates that the Ca2+-myristoyl switch can function with different lipid moieties and is no t strictly restricted to myristate. The ability to modify at will the fatty acid linked to the N-terminal glycine should be useful to analyze the cont ribution of the fatty acid moiety to the biological function of this family of neuronal calcium sensors,