The four calcium binding domains of calmodulin were synthesized using automated peptide synthesizer along with some variants of low and high affinity calcium binding sites. The calcium binding to various peptides were examined by binding of a cationic carbocyanin dye, named """"""""Stains- all"""""""". This dye has been shown to bind to calcium binding domains of various calcium binding proteins and generate specific absorption peaks and circular dichroism signals which are characteristic of the binding of this dye to calcium binding domains. Individual calcium binding domains of calmodulin binds Stains-all and induce only J band (near 620 nm) in the circular dichroism spectrum. Higher affinity calcium binding sites induce higher ellipticity of J band than lower affinity calcium binding sites when bound to Stains-all. Site I has lowest binding affinity and Site IV has highest binding affinity to the dye. Replacement of aspartic acid with asparagine in the + X position (of the octahedon coordinating the calcium ion) of the weaker calcium binding site (Site I) completely abolishes the dye binding while the same change in the higher affinity calcium binding site (Site IV) only reduces this dye binding but does not abolish it. Replacement of tyrosine in site IV with tryptophan does not distort the geometry of calcium binding sites but increases the dye binding. The mechanism by which this occurs requires further investigation. The binding of the dye to all calcium binding sites in calmodulin is completely displaced by calcium. However, more calcium is required to displace the dye from Site IV than from Site I.
