Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Steady-State Fluorescence Spectroscopy 85
b. Quenching studies with several quenching agents to obtain K SV, and then
repeat the quenching experiments at several Ca 2+ ion concentrations to obtain
a plot of K SV vs [Ca 2+ ]. If the calcium-binding protein interacts with another
species containing a fluorophore, such as a tryptophan containing protein/
peptide, or lanthanide ion:
c. Repeat the excitation and emission spectra and quenching experiments with
all species varied (e.g., protein alone, peptide alone, protein + Ca 2+ , peptide +
Ca 2+ , protein + peptide, protein + peptide + Ca 2+ ). If the secondary binding
species is a lanthanide ion that binds in the Ca 2+ -binding region of the protein,
then fluorescence of both the protein and the ion can be monitored in a
competitive titration experiment.
d. Red-edge emission spectral effect (REES) spectroscopy can be used to monitor
different populations of Trp (14), and determining if this is a sensitive tool
for following Ca 2+ -binding in titrations.
e. Fluorescence energy transfer (or Förster’s resonance effect spectroscopy,
FRET) can be used to monitor the distance between two specially chosen
fluorophores between 10–75 Å apart (3). There is a high possibility that
labeling with an extrinsic fluorophore will be necessary (5). Once a suitable
system is established, quenching experiments can again be used to determine
the relative solvent exposures of the two fluorophores, with and without Ca 2+ .
f. Fluorescence anisotropy is another tool used to study the interaction between
two biomolecules, and has successfully been applied to calcium binding systems
(4).
10. A selected set of examples that encompass major fluorescence applications to
calcium-binding proteins is given in Table 1. Many of these applications are
dependent on specific fluorophores (e.g., unique Trp residues, or attachment sites
for extrinsic fluorophores). The methods described in Table 1 also often require
instrumentation and expertise beyond the scope of this chapter, but are provided
for a demonstration of the usefulness of this technique.
11. Tb 3+ is a popular fluorescent Ca 2+ analogue, which can be used both intrinsically
as an indicator for metal ion-binding, Ca 2+ -binding competition experiments, and
also as part of a donor/acceptor pair in resonance transfer analysis. Caution should
be used in assessing structure–function relationships of proteins with bound Tb 3+
however, as protein activity can be effected by this substitution.
12. Mutagenesis studies in which Tyr/Phe residues are replaced by Trp for fluorescence
have been reported. It has been shown however that in the case of troponin
C, such mutagenesis alters the Ca 2+ -binding properties of the protein, and results
using such mutants must therefore be carefully evaluated (22,23).
Acknowledgments
We would like to offer our sincere gratitude to R. J. Turner for insightful discussions
and suggestions. A. M. Weljie would like to thank the National Sciences
and Engineering Research Council and the Alberta Heritage Fund for Medical
Research (AHFMR) for support. Also, H. J. Vogel is an AHFMR Scientist.