SECONDARY STRUCTURE AND CA2-INDUCED CONFORMATIONAL CHANGE OF CALEXCITIN, A LEARNING-ASSOCIATED PROTEIN()

Calexcitin/cp20 is a low molecular weight GTP- and Ca2+-binding protei n, which is phosphorylated by protein kinase C during associative lear ning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons, Here, the secondary s tructure of cloned squid calexcitin was determined by circular dichroi sm 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 sec ondary structure computed from the amino acid sequence, In solution, c alexcitin is one-third in alpha-helix and one-fifth in beta-sheet. The conformation of the protein in solid state depends on the concentrati on of the starting solution, suggesting the occurrence of surface aggr egation. The secondary structure also depends on the binding of calciu m, which causes an increase in alpha-helix and a decrease in beta-shee t, as estimated by circular dichroism, The conformation of calexcitin is independent of ionic strength, and the calcium-induced structural t ransition is slightly inhibited by Mg2+ and low pH, while favored by h igh pH. The switch of calexcitin's secondary structure upon calcium bi nding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physi ologically meaningful range of Ca2+ concentration. The calcium-bound f orm is more globular than the apoprotein, Unlike other EF-hand protein s, calexcitin's overall lipophilicity is not affected by calcium bindi ng, as assessed by hydrophobic liquid chromatography. Preliminary resu lts from patch-clamp experiments indicated that calcium is necessary f or calexcitin to inhibit potassium channels and thus to increase membr ane excitability, Therefore the calcium-dependent conformational equil ibrium of calexcitin could serve as a molecular switch for the short t erm modulation of neuronal activity following associative conditioning .