title: Secondary structure and Ca2+-induced conformational change of calexcitin, a learning-associated protein.
abstract: 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 alpha-helix and one-fifth in beta-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 alpha-helix and a decrease in beta-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.
authors.affiliation: Laboratory of Adaptive Systems, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA. ascoli@nih.gov
authors.forename: G A
authors.initials: GA
authors.lastname: Ascoli
authors.forename: K X
authors.initials: KX
authors.lastname: Luu
authors.forename: J L
authors.initials: JL
authors.lastname: Olds
authors.forename: T J
authors.initials: TJ
authors.lastname: Nelson
authors.forename: P A
authors.initials: PA
authors.lastname: Gusev
authors.forename: C
authors.initials: C
authors.lastname: Bertucci
authors.forename: E
authors.initials: E
authors.lastname: Bramanti
authors.forename: A
authors.initials: A
authors.lastname: Raffaelli
authors.forename: P
authors.initials: P
authors.lastname: Salvadori
authors.forename: D L
authors.initials: DL
authors.lastname: Alkon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D049832:Decapodiformes; D004533:Egtazic Acid / Q000494:pharmacology; D005347:Fibroblasts; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007858:Learning; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009420:Nervous System / Q000737:chemistry; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011487:Protein Conformation / Q000187:drug effects*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000494:pharmacology; D012879:Skin Physiological Phenomena; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D015221:Potassium Channels; D011994:Recombinant Proteins; D004533:Egtazic Acid; D019204:GTP-Binding Proteins; C102751:cex-2 protein, C elegans; D002118:Calcium