The four calcium binding domains of calmodulin were synthesized using an automated peptide synthesizer. Calcium binding to these calmodulin fragments were examined by using high resolution 2D NMR. The configuration and structural dynamics of the synthetic calmodulin fragments have been studied both in the presence and absence of calcium using 2D correlation spectroscopies such as COSY (Chemical Shift Correlation), NOSEY (Nuclear Overhauser Effect Spectroscopy) and HOHAH (Homonuclear Hartman Hahn Spectroscopy). Extensive one dimensional and two dimensional correlation spectroscopy has been performed on all four calcium binding sites of calmodulin. Detailed investigations have been completed for the fragment representing the 4th calcium binding domain in calmodulin, which represents the high affinity calcium binding site. The calcium binding region in this peptide adopts a random structure in the absence of calcium. On the addition of calcium, this region adopts a rigid structure as indicated by the appearance of space NOE coupling in the NMR. The 16 amino acids preceding the calcium binding region adopt a random coil structure in the absence of calcium, but in the presence of calcium, these amono acids adopt an alpha helical structure as indicated by 2D NH-NH NOSEY NMR spectroscopy.
