Determination of the metal-binding cooperativity of wild-type and mutant calbindin D9K by electrospray ionization mass spectrometry.
Since the initial reports showing the ability of electrospray ionization mass spectrometry (ESI-MS) to study intact noncovalent biomolecular complexes, an increasing number of uses for this technique in studying biochemical systems is emerging. We have investigated the ability of ESI-MS to characterize the metal-binding properties of calcium (Ca2+) binding proteins by studying the incorporation of Ca2+ and cadmium (Cd2+) into wild-type and mutant calbindin D9K. ESI-MS showed that wild-type calbindin D9K binds two Ca2+ ions with similar affinities while the binding of two Cd2+ ions is sequential, as is the binding of the two Ca2+ or Cd2+ ions to the N56A mutant of calbindin. 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 metalloproteins.
Chazin
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Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Veenstra
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TD
D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D064026:Calbindins; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D064026:Calbindins; D008670:Metals; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; D002104:Cadmium; D002118:Calcium