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Eur. J. Mass Spectrom. 3, 453 - 459 (1997)

Electrospray ionization mass spectrometry temperature effects on metal ion:protein stoichiometries and metal-induced conformational changes in calmodulin

Timothy D. Veenstra
Department of Nephrology Research Unit, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
Kenneth L. Johnson and Andy J. Tomlinson
Biomedical Mass Spectrometry Facility and Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
Stephen Naylor*
Biomedical Mass Spectrometry Facility, Department of Biochemistry and Molecular Biology and Department of Pharmacology and Clinical Pharmacological Unit, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
Rajiv Kumar*
Department of Nephrology Research Unit and Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT:
The calcium ion (Ca2+) binding stoichiometry required to induce a complete, tertiary conformational change of calmodulin is still disputed. Several studies have indicated that this occurs upon the uptake of only two Ca2+; more recent reports, however, indicate that four Ca2+ are required. We used electrospray ionization (ESI) mass spectrometry under standard ESI conditions (i.e. high temperature, organic co-solvent) to identify definitively the Ca2+ stoichiometry required to induce a conformational change in the protein, and we demonstrate that four Ca2+ are needed. We then undertook a comparative study on the Ca2+-binding of calmodulin using a lower ESI source temperature. Under these conditions we can detect Ca2+-saturated calmodulin at much lower Ca2+ to protein molar ratios than those observed using typical ESI conditions. This latter observation more closely reflects the solution-phase conditions that are noted using aqueous solution-based spectroscopies to study proteinmetal binding.

Keywords: Low temperature, electrospray ionization mass spectrometry, calmodulin, calcium, conformation.

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