Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Spatial Distribution of Ca 2+ -Binding Proteins 405
Fig. 17. Indicator of calmodulin activation using TA-calmodulin and FLcalmodulin.
Simultaneous use of the activation-sensitive and activation-insensitive
derivative allows us to distinguish between activation and local concentration changes
of calmodulin. The sequence shows mitotic events from 79–121 min.
FL-calmodulin is an inert fluorophore. If the [Ca 2+ ] is changed from 10 nM – 0.1 mM
at physiological ionic strength and pH, the fluorescence intensity of FL-calmodulin
changes by less than 5%. No further change occurs on peptide target binding.
Similar observations were made with Cy5-calmodulin and Texas Red-calmodulin.
Thus FL-, Cy5-, and Texas Red-calmodulins report calmodulin localization in
the cell. In contrast, if calmodulin is labeled at Lys 75 with the environmentally
sensitive TA-Cl probe (11), Ca 2+ -binding results in a 5.5-fold increase of fluorescence
intensity and target binding may cause a further twofold increase (12).
TA-calmodulin fluorescence thus reports the interactions of calmodulin, as well
as its concentration changes in the cell. In order to distinguish between the two
events, TA- and FL-calmodulin can both be applied to the same cell and their
fluorescence emissions are scanned simultaneously.
5. It is instructive to compare the target binding and enzyme activation properties of
fluorescently labeled calmodulin with unlabeled calmodulin. TA-calmodulin and