Calcium and Membrane Binding Properties of Bovine Neurocalcin 
Expressed in Escherichia coli(*)
- From the Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305
Abstract
Neurocalcins are brain-specific proteins that belong to a new subclass of the EF-hand superfamily of calcium binding proteins,
defined by the photoreceptor cell-specific protein, recoverin. Recoverin, which regulates the desensitization of photo-excited
rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch. Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first one lacks some residues critical for calcium binding. In this
work, I have examined the calcium and membrane binding properties of recombinant myristoylated and unmyristoylated neurocalcin
. I show that neurocalcin, like recoverin, binds to biological membranes in a calcium- and myristoyl-dependent manner. Both
myristoylated and unmyristoylated proteins bind three calcium ions. However, the unmyristoylated form exhibits a higher affinity
for calcium than the myristoylated protein but shows a lower cooperativity in binding calcium. These data support the model
for the calcium-myristoyl switch mechanism proposed for recoverin (Zozulya, S., and Stryer, L.(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11569-11573; Dizhoor, A. M., Chen, C. K., Olshevskaya, E., Sinelnikova, V. V., and Hurley, J. B. (1993) Science 259, 829-832). Using point mutations, I have investigated the relative importance of each of the three functional EF hands
(EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin. Calcium and membrane binding properties
of the mutant-myristoylated proteins suggest that binding of calcium to EF2 is critical in triggering the binding of the protein
to membranes.
Footnotes
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↵§ Supported by a fellowship from CNRS/National Science Foundation. Present address: Département de Biochimie et Génètique Moléculaire, Institut Pasteur, 28, rue du Docteur Roux, F-75 724 Paris-Cedex 15, France. Tel.: 33-1-45-68-83-84; Fax: 33-1-40-61-30-19; Ladant{at}pasteur.fr
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↵* This work was supported by National Institutes of Health Grants MH45324 and GM24032 (to L. Stryer). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- MOPS
-
4-morpholinepropanesulfonic acid
- ME84Q
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ME120Q, and ME168Q, protein designations for myristoylated glutamic acid to glutamine-mutated neurocalcin.
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↵2 D. Ladant, S. Zozulya, and L. Stryer, unpublished observations.
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- Received October 31, 1994.
- Revision received December 1, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
