Analysis of the ion binding sites of calmodulin by electrospray ionization mass spectrometry.

The binding of Ca2+ and Mg2+ to four calmodulins (SynCaM 1, SynCaM 8, SynCaM 12A, and SynCaM 18A) has been studied by ESI-MS. The mass spectra were recorded by dissolving the apoproteins in methanol/water (20/80, v/v) containing 1 mM CaCl2 or 1 mM MgCl2 and the pH adjusted to 6.0 with ammonia. The carrier solvent was methanol/water (20/80, v/v). In the case of Ca2+ complexation, ESI-MS reveals the presence of three kinds of sites: the first of high affinity corresponding to those determined using flow and equilibrium dialysis techniques and two others with lower affinities. These results clearly confirm the conclusion of Milos et al. [Milos, M., Comte, M., Schaer, J. J., & Cox, J. A. (1989) J. Inorg. Biochem. 36, 11-25] that there should exist between four and six auxiliary sites for Ca2+. Concerning the complexation of magnesium, the four proteins are able to bind two Mg2+ almost certainly on auxiliary cationic sites.