Correlation of fluorescence and circular dichroism spectroscopy with electrospray ionization mass spectrometry in the determination of tertiary conformational changes in calcium-binding proteins

Timothy D. Veenstra, Kenneth L. Johnson, Andy J. Tomlinson, Rajiv Kumar, Stephen Naylor

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We compared changes in the fluorescence, circular dichroism (CD) and multiply charged electrospray ionization mass spectrometry (ESI-MS) spectra of three calcium (Ca2+)-binding proteins upon the binding of Ca+. The proteins used were rat brain calbindin D(28K) and two deletion mutants, one lacking EF-hand 2 (calbindin Δ2) and the other lacking EF-hands 2 and 6 (calbindin Δ2,6). Large changes in the intrinsic protein fluorescence spectrum were seen upon the addition of Ca2+ to calbindin D(28K) and Δ2, while a less significant change was observed for calbindin Δ2,6. In a fluorescent study in which p-toluidinyl-2-naphthalene-6-sulfonote, a fluorescent probe which binds to hydrophobic surfaces within proteins, was used; calbindin D(28K) and Δ2 again showed a greater change in fluorescence intensity upon Ca2+-binding than calbindin Δ2,6. Near UV-CD studies, which measure changes within the tertiary structure of a protein, showed greater changes in the spectrum of calbindin D(28K) and Δ2 compared to calbindin Δ2,6 upon Ca2+-binding. Far UV-CD studies, which measures changes within the secondary structure of a protein, however, showed that the spectrum of all three proteins underwent only minor changes upon metal-binding. The ESI-MS studies showed that as the proteins were titrated with Ca2+ a gradual shift in the mass envelope from higher to lower charge states occurs. In the case of calbindin D(28K) and calbindin Δ2, however, a complete shift in the mass envelope towards the lower charge states is observed upon saturation with Ca2+, whereas for calbindin Δ2,6, the shift in the charge states is still relatively evenly distributed between high and low charge states. Changes within the ESI-MS spectrum observed upon the addition of Ca2+ correlated with Ca2+-induced changes observed with near-ultraviolet CD, intrinsic fluorescence spectroscopy, and spectroscopy using the fluorescent probe. Changes in the far ultraviolet-CD spectra of the calbindins, however, did not correlate with changes in the ESI-MS spectra upon calcium binding. The results show that ESI-MS can be use to detect changes in the tertiary structure of calcium-binding proteins induced by the binding of metal to the proteins.

Original languageEnglish (US)
Pages (from-to)613-619
Number of pages7
JournalRapid Communications in Mass Spectrometry
Issue number10
StatePublished - 1998


ASJC Scopus subject areas

  • Analytical Chemistry
  • Spectroscopy

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