Pharmaceutical Sciences Faculty Publications

Correlation of Fluorescence and Circular Dichroism Spectroscopy with Electrospray Ionization Mass Spectrometry in the Determination of Tertiary Conformational Changes in Calcium‐Binding Proteins

Document Type


Publication Date


Journal Title

Rapid Communications in Mass Spectrometry





First Page


Last Page





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 D28K and two deletion mutants, one lacking EF‐hand 2 (calbindin &Dgr;2) and the other lacking EF‐hands 2 and 6 (calbindin &Dgr;2,6). Large changes in the intrinsic protein fluorescence spectrum were seen upon the addition of Ca2+ to calbindin D28K and &Dgr;2, while a less significant change was observed for calbindin &Dgr;2,6. In a fluorescent study in which &rgr;‐toluidinyl‐2‐naphthalene‐6‐sulfonate, a fluorescent probe which binds to hydrophobic surfaces within proteins, was used; calbindin D28K and &Dgr;2 again showed a greater change in fluorescence intensity upon Ca2+‐binding than calbindin &Dgr;2,6. Near UV‐CD studies, which measure changes within the tertiary structure of a protein, showed greater changes in the spectrum of calbindin D28K and &Dgr;2 compared to calbindin &Dgr;2,6 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 D28K and calbindin &Dgr;2, however, a complete shift in the mass envelope towards the lower charge states is observed upon saturation with Ca2+, whereas for calbindin &Dgr;2,6, 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. © 1998 John Wiley & Sons, Ltd.


Mass spectrometry, electrospray ionization, florescence, circular dichroism, tertiary, calcium-binding proteins