Premium
Determination of the metal‐binding cooperativity of wild‐type and mutant calbindin D 9K by electrospray ionization mass spectrometry
Author(s) -
Chazin Walter,
Veenstra Timothy D.
Publication year - 1999
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19990330)13:6<548::aid-rcm523>3.0.co;2-u
Subject(s) - chemistry , electrospray ionization , cooperativity , mutant , mass spectrometry , electrospray , chromatography , biochemistry , gene
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 (Ca 2+ ) binding proteins by studying the incorporation of Ca 2+ and cadmium (Cd 2+ ) into wild‐type and mutant calbindin D 9K . ESI‐MS showed that wild‐type calbindin D 9K binds two Ca 2+ ions with similar affinities while the binding of two Cd 2+ ions is sequential, as is the binding of the two Ca 2+ or Cd 2+ ions to the N56A mutant of calbindin. The binding of Ca 2+ 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. Copyright © 1999 John Wiley & Sons, Ltd.