Static and Kinetic Studies of Complex Formations between Calmodulin and MastoparanX

Ca2+-induced complex formation between calmodulin (CaM) and mastoparanX (MasX) was studied by a fluorescence spectroscopy and by a stopped-flow method. The measurements of the fluorescence anisotropy in the presence of calcium and the fluorescence titration with Ca2+ revealed that the N- and C-domains of CaM bound cooperatively MasX, while the tryptic fragments of CaM (TR1C, 1−77 and TR2C, 78−148) bound independently MasX. The Trp-fluorescence stopped-flow experiments revealed that the Ca2+-induced binding of CaM and MasX was composed of two processes: one was a rapid binding of the N-domain of CaM to MasX, which was induced by the rapid Ca2+ binding to the N-sites of CaM. The other was a slow biphasic process. Its fast phase was the binding of the C-domain of CaM to MasX, which was induced by the slow Ca2+ binding to the C-sites. Interestingly, the kinetics of the slow process varied with the Ca2+ concentrations. At the low Ca2+ concentrations, its rate constant increased to around 20 s-1 as the Ca2+ concentration increased. At the high Ca2+ concentrations, the Ca2+-induced binding of the C-domain of CaM to MasX proceeded at a constant rate around 20 s-1. This suggested an existence of a rate-limiting step for the Ca2+-induced binding of the C-domain of CaM to MasX at the high Ca2+ concentrations. The slow phase of the slow process may be a rearrangement of the CaM−MasX complex. These results led to our model of a molecular kinetic mechanism of the Ca2+-induced complex formation between CaM and MasX.