DETERMINING CALCIUM-BINDING STOICHIOMETRY AND COOPERATIVITY OF PARVALBUMIN AND CALMODULIN BY MASS-SPECTROMETRY

The calcium-binding properties of parvalbumin and calmodulin were inve stigated by electrospray ionization mass spectrometry (ESI-MS). The tw o calcium sites of parvalbumin were found to be strongly cooperative i n binding Ca2+ ion. Up to four calcium ions were found to bind to calm odulin at high calcium concentration levels. Strong cooperativity was detected between the third and fourth Ca2+ binding sites of calmodulin (ordered by loading sequence). Strong interactions were also indicate d between the two halves of the calmodulin molecule. Demetallation of the gas-phase ions can occur during the desolvation process, especiall y for ESI of aqueous solutions. However, the ESI-MS methodology has th e potential to provide insight into the intricate processes involved i n metal site communications for other metalloproteins.