Pharmaceutical Sciences Faculty Publications

Determination of the Metal-Binding Cooperativity of Wild-Type and Mutant Calbindin D9K by Electrospray Ionization Mass Spectrometry

Document Type

Article

Publication Date

1-1-1999

Journal Title

Rapid Communications in Mass Spectrometry

ISSN

0951-4198

Volume

13

Issue

6

First Page

548

Last Page

555

DOI

10.1002/(SICI)1097-0231(19990330)13:6<548::AID-RCM523>3.0.CO;2-U

PubMed ID

10204248

Abstract

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.

Keywords

Cadmium, calbindins, calcium, mass spectrometry, metals, mutation, nerve tissue proteins, protein binding

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