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The synthesis of the cyclic 20-31 sequence of bovine brain calmodulin corresponding to the loop of the hypothetical calcium binding site I of the protein has been accomplished by classical solution methods. The interaction of the synthetic cyclic fragment with calcium ions has been investigated by CD spectroscopy in water and in 98% trifluoroethanol solution. Calcium ions have no effect on the dichroic absorption of aqueous solution of the cyclic dodecapeptide in the wavelength range 200-250 nm. In 98% trifluoroethanol the CD spectrum of the cyclic compound in the absence of calcium ions is almost identical to that of the linear dodecapeptide in the presence of saturation concentrations of calcium. This result supports our previous hypothesis of a folding of the linear sequence upon interaction with the metal ion. The cyclic peptide also interacts with calcium ions in 98% trifluoroethanol forming a 1:1 complex.

The synthesis of the dodecapeptide [sequence (20--31)] representing the hypothetical calcium binding site I of calmodulin by classical methods in solution is described. The interaction of this synthetic calmodulin fragment with calcium ions has been investigated by CD in water and in 98% trifluoroethanol solution. Calcium ions have no effect on the dichroic absorption of the dodecapeptide in aqueous solution in the wavelength range 200-250 nm. However, in 98% trifluoroethanol a linear variation of the CD properties has been observed as a function of the Ca2/peptide molar ratio. The CD results indicate the formation of a 1 : 1 calcium-dodecapeptide complex with a very high binding constant.

We report details of the chemical synthesis of the dodecapeptide corresponding to the calcium binding loop III of bovine brain calmodulin (sequence 93-104) and its fragments 96-04, 93-98, and 99-104. The preparation of the peptides employed classical solution methods and a fragment-condensation strategy. The major difficulties were encountered during the synthesis of the peptides containing the N-terminal sequences -Gly-Asn-Gly- and -Asp-Lys-Asp-Gly-Ans-Gly-, in which alpha-beta shift side reactions were observed.

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.