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Volume 272, Number 40, Issue of October 3, 1997 pp. 24771-24779
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Secondary Structure and Ca²⁺-induced Conformational Change of Calexcitin, a Learning-associated Protein

(Received for publication, April 10, 1997, and in revised form, June 9, 1997)

Giorgio A. Ascoli ^§ , Kieu X. Luu , James L. Olds , Thomas J. Nelson , Pavel A. Gusev , Carlo Bertucci ^§ , Emilia Bramanti , Andrea Raffaelli ^§ , Piero Salvadori ^§ and Daniel L. Alkon

From the  Laboratory of Adaptive Systems, NINDS, National Institutes of Health, Bethesda, Maryland 20892, ^§ Centro di Studio per le Macromolecole Stereordinate ed Otticamente Attive del C.N.R., Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Risorgimento 35, 56126 Pisa, Italy, and  Institute of Instrumental Analytical Chemistry, C.N.R., Via Risorgimento 35, 56126 Pisa, Italy

Calexcitin/cp20 is a low molecular weight GTP- and Ca²⁺-binding protein, which is phosphorylated by protein kinase C during associative learning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons. Here, the secondary structure of cloned squid calexcitin was determined by circular dichroism in aqueous solution and by Fourier transform infrared spectroscopy both in solution and on dried films. The results obtained with the two techniques are in agreement with each other and coincide with the secondary structure computed from the amino acid sequence. In solution, calexcitin is one-third in -helix and one-fifth in -sheet. The conformation of the protein in solid state depends on the concentration of the starting solution, suggesting the occurrence of surface aggregation. The secondary structure also depends on the binding of calcium, which causes an increase in -helix and a decrease in -sheet, as estimated by circular dichroism. The conformation of calexcitin is independent of ionic strength, and the calcium-induced structural transition is slightly inhibited by Mg²⁺ and low pH, while favored by high pH. The switch of calexcitin's secondary structure upon calcium binding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physiologically meaningful range of Ca²⁺ concentration. The calcium-bound form is more globular than the apoprotein. Unlike other EF-hand proteins, calexcitin's overall lipophilicity is not affected by calcium binding, as assessed by hydrophobic liquid chromatography. Preliminary results from patch-clamp experiments indicated that calcium is necessary for calexcitin to inhibit potassium channels and thus to increase membrane excitability. Therefore the calcium-dependent conformational equilibrium of calexcitin could serve as a molecular switch for the short term modulation of neuronal activity following associative conditioning.

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