Correlation of fluorescence and circular dichroism spectroscopy with electrospray ionization mass spectrometry in the determination of tertiary conformational changes in calcium-binding proteins
✍ Scribed by Timothy D. Veenstra; Kenneth L. Johnson; Andy J. Tomlinson; Rajiv Kumar; Stephen Naylor
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 173 KB
- Volume
- 12
- Category
- Article
- ISSN
- 0951-4198
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✦ Synopsis
We compared changes in the fluorescence, circular dichroism (CD) and multiply charged electrospray ionization mass spectrometry (ESI-MS) spectra of three calcium (Ca 2 )-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 D2) and the other lacking EF-hands 2 and 6 (calbindin D2,6). Large changes in the intrinsic protein fluorescence spectrum were seen upon the addition of Ca 2 to calbindin D 28K and D2, while a less significant change was observed for calbindin D2,6. In a fluorescent study in which &-toluidinyl-2naphthalene-6-sulfonate, a fluorescent probe which binds to hydrophobic surfaces within proteins, was used; calbindin D 28K and D2 again showed a greater change in fluorescence intensity upon Ca 2 -binding than calbindin D2,6. 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 D2 compared to calbindin D2,6 upon Ca 2binding. 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 Ca 2 a gradual shift in the mass envelope from higher to lower charge states occurs. In the case of calbindin D 28K and calbindin D2, however, a complete shift in the mass envelope towards the lower charge states is observed upon saturation with Ca 2 , whereas for calbindin D2,6, 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 Ca 2 correlated with Ca 2 -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.