ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY TEMPERATURE EFFECTS ON METAL-ION - PROTEIN STOICHIOMETRIES AND METAL-INDUCED CONFORMATIONAL-CHANGES IN CALMODULIN

The calcium ion (Ca2+) binding stoichiometry required to induce a comp lete, tertiary conformational change of calmodulin is still disputed. Several studies hale indicated that this occurs upon the uptake of onl y two Ca2+; more recent reports, however, indicate that four Ca2+ are required. We used electrospray ionization (ESI) mass spectrometry unde r standard ESI conditions (i.e. high temperature, organic co-solvent) to identify definitively the Ca2+ stoichiometry required to induce a c onformational change in the protein, and we demonstrate that four Ca2 are needed. We then undertook a comparative study on the Ca2+-binding of calmodulin using a lower ESI source temperature. Under these condi tions we can detect Ca2+-saturated calmodulin at much lower Ca2+ to pr otein molar ratios than those observed using typical ESI conditions, T his latter observation more closely reflects the solution-phase condit ions that are noted using aqueous solution-based spectroscopies to stu dy protein-metal binding.