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 Ca(2+)-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 as "Ca(2+)-myristoyl switch" has been best described for the photoreceptor specific protein, recoverin, as well as for several other neuronal calcium sensors. Here, we used reversed micelles to chemically acylate nonmyristoylated neurocalcin at its N-terminus with fatty acids of different lengths (from C12 to C16). This approach allowed us to prepare neurocalcin derivatives in which a single fatty acid is selectively linked to the N-terminal glycine of the polypeptide chain through an amide bond. The membrane binding properties of the monoacylated neurocalcins were then examined by cosedimentation with phospholipid vesicles and direct binding to lipid monolayers by surface plasmon resonance spectroscopy (Biacore). Our results show that neurocalcins monoacylated with lauric, myristic, or palmitic acid were able to associate with membrane in a calcium-dependent manner. This indicates that the Ca(2+)-myristoyl switch can function with different lipid moieties and is not 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 contribution of the fatty acid moiety to the biological function of this family of neuronal calcium sensors.
Béven
L
L
UMR 6022 CNRS, Université de Technologie de Compiègne, BP 20529, 60205 Compiègne Cedex, France.
Adenier
H
H
Kichenama
R
R
Homand
J
J
Redeker
V
V
Le Caer
J P
JP
Ladant
D
D
Chopineau
J
J
D000215:Acylation; D000818:Animals; D015374:Biosensing Techniques; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D008823:Micelles; D019814:Myristic Acid / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051596:Neurocalcin; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D044169:Receptors, Calcium-Sensing; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002135:Calcium-Binding Proteins; D008081:Liposomes; D008823:Micelles; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D010446:Peptide Fragments; D010743:Phospholipids; D044169:Receptors, Calcium-Sensing; D019814:Myristic Acid; D002118:Calcium