Cat - Dependent Changes in Helical Structure of Calmodulin

Structure of calmodulin in solution was studied by measurements of the far UV CD spectrum. The CD spectrum of Ca?+-saturated calmodulin obtained at pH 7.0 was superimposed on the one at pH 5.6. Therefore, the structure of calmodulin at pH 7.0 may be identical to the crystalline structure determined at pH 5.6.1) The of-helix content of Ca`'+-free calmodulin was 18% lower than that of CaJ+-saturated form and was decreased by further 19% when NaCI was removed from the solution. The NaCI-dependent changes in a-helix content of Ca'+-free calmodulin was not remarkable after tryptic cleavage at the middle of the central a-helix connecting Nand C-domains. Positive charges are concentrated around the trypsin sensitive region and the charges may be neutralized by NaCI, which may function to maintain the helical structure of central rod. Two residues with a positive charge are eliminated by the proteolysis which may release the repulsive force. The latent flexibility of the central a-helix may be important for the multiple target activation mechanisms of calmodulin. Introduction. In 1982 Drabikowski et al. established a tryptic digestion method to yield Nand C-terminal halves of the calmodulin molecule.9) Minowa and Yagi3) purified each fragment of scallop calmodulin designated here as F12 and F34, respectively. Both F12 and F34 retained 2 Ca?+ binding sites with the affinities indistinguishable from those in calmodulin. Ikura et al. reported on the basis of measurements of 111 NMR spectrum that simple addition of the spectra of two fragments yielded the spectrum of calmodulin.4~ Therefore, it has been indicated that the tertiary structures of the fragments F12 and F34 were apparently identical to those in calmodulin. These results were well explained by the three dimensional structure of calmodulinl~ in which Nand C-domains, corresponding to F12 and F34, respectively, were separated with a single 8 turn a-helix. On the other hand, Minowa et al.>> reported that isolated fragments F12, F34 or their mixture could not activate the target enzymes. In the present study we compared the Cac+-dependent change in the helical structure of calmodulin with those of fragments. We found a clear difference in the helical structures between calmodulin and a mixture of the fragments. The difference suggests a significance of the a-helical region in the interaction of calmodulin with target proteins. Materials and methods. Preparation of scallop calmodulin and its tryptic fragments, F12 (1-75) and F34 (78-148), were described.3> CD spectrum was measured using a JASCO J500A spectropolarimeter equipped with a data pro-