Determination of the metal-binding cooperativity of wild-type and mutant calbindin D-9K by electrospray ionization mass spectrometry

Since the initial reports showing the ability of electrospray ionization ma ss spectrometry (ESI-MS) to study intact noncovalent biomolecular complexes , an increasing number of uses for this technique in studying biochemical s ystems is emerging. We have investigated the ability of ESI-MS to character ize the metal-binding properties of calcium (Ca2+) binding proteins by stud ying the incorporation of Ca2+ and cadmium (Cd2+) into wild-type and mutant calbindin D-9K. ESI-MS showed that wild-type calbindin D-9K binds two Ca2 ions with similar affinities while the binding of two Cd2+ ions is sequent ial, as is the binding of the two Ca2+ or Cd2+ ions to the N56A mutant of c albindin. The binding of Ca2+ to the wild-type protein was clearly seen to be cooperative. These results demonstrate the potential efficacy of ESI-MS to discriminate between cooperative and independent site metal binding to m etalloproteins. Copyright (C) 1999 John Wiley & Sons, Ltd.