Determination of the metal-binding cooperativity of wild-type and mutant calbindin D9K by electrospray ionization mass spectrometry

✍ Scribed by Walter Chazin; Timothy D. Veenstra

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 96 KB
• Volume: 13
• Category: Article
• ISSN: 0951-4198
• DOI: 10.1002/(sici)1097-0231(19990330)13:6<548::aid-rcm523>3.0.co;2-u

✦ Synopsis

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 metalbinding 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.