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Correlation of fluorescence and circular dichroism spectroscopy with electrospray ionization mass spectrometry in the determination of tertiary conformational changes in calcium‐binding proteins
Author(s) -
Veenstra Timothy D.,
Johnson Kenneth L.,
Tomlinson Andy J.,
Kumar Rajiv,
Naylor Stephen
Publication year - 1998
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19980529)12:10<613::aid-rcm202>3.0.co;2-5
Subject(s) - chemistry , circular dichroism , calcium binding protein , electrospray ionization , fluorescence , calbindin , mass spectrometry , protein secondary structure , biophysics , calcium , crystallography , analytical chemistry (journal) , biochemistry , chromatography , physics , organic chemistry , quantum mechanics , biology
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 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 Ca 2+ to calbindin D 28K and Δ2, while a less significant change was observed for calbindin Δ2,6. In a fluorescent study in which ρ‐toluidinyl‐2‐naphthalene‐6‐sulfonate, 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 Ca 2+ ‐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 Ca 2+ ‐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 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 Δ2, however, a complete shift in the mass envelope towards the lower charge states is observed upon saturation with Ca 2+ , 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 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. © 1998 John Wiley & Sons, Ltd.