Calcium-induced conformational changes of the recombinant CBP3 protein from Dictyostelium discoideum.

Calcium-binding proteins play various and significant roles in biological systems. Conformational changes in their structures are closely related to their physiological functions. To understand the role of calcium-binding protein 3 (CBP3) in Dictyostelium discoideum, its recombinant proteins were analyzed using circular dichroism (CD) and fluorescence spectroscopy. Gel mobility shift analysis showed that Ca2+ induced a mobility shift of the recombinant CBP3. Far ultra-violet CD spectra and intrinsic fluorescence spectra on CBP3 and its N- and C-terminal domains exhibited that they underwent a conformational rearrangement depending upon Ca2+ binding. Measurement of Ca2+ dissociation constants demonstrated that CBP3 had high affinity toward Ca2+ in the sub-micromolar range and N-terminal domain had higher affinity than C-terminal domain. The changes of fluorescence spectra by an addition of 8-anilino-1-naphthalene sulfonic acid indicated that the hydrophobic patches of CBP3 and its C-terminal domain are likely to be more exposed in the presence of Ca2+. Since the exposure of hydrophobic patches is thermodynamically unfavorable, Ca2+-bound CBP3 may interact with other proteins in vivo. All these data suggest that Ca2+ induces CBP3 to be more favorable conformation to interact with target proteins.

[1]  Y. Maeda,et al.  Specific expression of a gene encoding a novel calcium‐binding protein, CAF‐1, during transition of Dictyostelium cells from growth to differentiation , 1995, Development, growth & differentiation.

[2]  J. Williams,et al.  Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis. , 1992, Molecular biology of the cell.

[3]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[4]  N. Sreerama,et al.  A self-consistent method for the analysis of protein secondary structure from circular dichroism. , 1993, Analytical biochemistry.

[5]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[6]  Charles E. Bugg,et al.  Three-dimensional structure of calmodulin , 1985, Nature.

[7]  J. Gergely,et al.  Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction. , 1984, CRC critical reviews in biochemistry.

[8]  K. Okamoto,et al.  Cytoplasmic Ca2+ and H+ concentrations determine cell fate in Dictyostelium discoideum , 1994, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  A. Pastore,et al.  Molecular mechanism of the calcium‐induced conformational change in the spectrin EF‐hands. , 1995, The EMBO journal.

[10]  C. Siu,et al.  Characterization of the cell adhesion molecule gp24 in Dictyostelium discoideum. Mediation of cell-cell adhesion via a Ca(2+)-dependent mechanism. , 1993, The Journal of biological chemistry.

[11]  E. Schmidt,et al.  Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum. , 1994, Journal of cell science.

[12]  B. André,et al.  Dictyostelium discoideum contains a family of calmodulin‐related EF‐hand proteins that are developmentally regulated , 1996, FEBS letters.

[13]  P. Fisher,et al.  Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx. , 1997, Journal of cell science.

[14]  Y. Maeda,et al.  The signals for starvation response are transduced through elevated [Ca2+]i in Dictyostelium cells. , 1998, Experimental cell research.

[15]  Christophe Geourjon,et al.  An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum , 1993, Comput. Appl. Biosci..

[16]  Ad Bax,et al.  Solution structure of calcium-free calmodulin , 1995, Nature Structural Biology.

[17]  V. Nanjundiah,et al.  Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA , 1994, FEBS letters.

[18]  B D Sykes,et al.  Role of the structural domain of troponin C in muscle regulation: NMR studies of Ca2+ binding and subsequent interactions with regions 1-40 and 96-115 of troponin I. , 2000, Biochemistry.

[19]  Mitsuhiko Ikura,et al.  Calcium-induced conformational transition revealed by the solution structure of apo calmodulin , 1995, Nature Structural Biology.

[20]  M Ikura,et al.  Molecular and structural basis of target recognition by calmodulin. , 1995, Annual review of biophysics and biomolecular structure.

[21]  A. B. Cubitt,et al.  Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin. , 1995, Development.

[22]  P. Fisher,et al.  A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium. , 1996, The EMBO journal.

[23]  A. Dizhoor,et al.  Role of the acylated amino terminus of recoverin in Ca(2+)-dependent membrane interaction. , 1993, Science.

[24]  D. Malchow,et al.  On the role of calcium during chemotactic signalling and differentiation of the cellular slime mould Dictyostelium discoideum. , 1996, The International journal of developmental biology.

[25]  A. Noegel,et al.  The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes. , 1995, Journal of cell science.

[26]  R. Kretsinger,et al.  Carp muscle calcium-binding protein. II. Structure determination and general description. , 1973, The Journal of biological chemistry.

[27]  V. Nanjundiah,et al.  A Ca(2+)-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry. , 1996, Experimental cell research.

[28]  Sa‐Ouk Kang,et al.  Cloning of a cDNA encoding a new calcium‐binding protein from Dictyostelium discoideum and its developmental regulation 1 , 1998 .

[29]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[30]  B. Coukell,et al.  Cloning and expression in Escherichia coli of a cDNA encoding a developmentally regulated Ca2+‐binding protein from Dictyostelium discoideum , 1995, FEBS letters.

[31]  M Ikura,et al.  Diversity of conformational states and changes within the EF‐hand protein superfamily , 1999, Proteins.

[32]  J. Enghild,et al.  Calbindin D28k Exhibits Properties Characteristic of a Ca2+ Sensor* , 2002, The Journal of Biological Chemistry.

[33]  S. Linse,et al.  Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k). , 2000, Biochemistry.

[34]  Eva Thulin,et al.  Calcium-induced structural changes and domain autonomy in calmodulin , 1995, Nature Structural Biology.