Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

90 Johnson and Tikunova
is removed from these sites with ethylene glycol-bis N,N,N',N'-tetraacetic acid
(EGTA). We use fluorescence stopped-flow techniques to measure the rates of
the fluorescence changes that occur upon Ca 2+ association and dissociation
from this protein.
The rates of Ca 2+ -induced structural (fluorescence) changes in Ca 2+ -binding
proteins may be slower than the actual rates of Ca 2+ binding and Ca 2+ dissociation.
For this reason, it is necessary to have a method of monitoring Ca 2+ off
rates, which is independent of structural changes in the protein. The fluorescent
Ca 2+ chelator, Quin-2, can be used to measure Ca 2+ off-rates from native
unlabeled proteins (7,8). In this technique, Quin is rapidly mixed with a Ca 2+ -
binding protein with bound Ca 2+ . Quin’s fluorescence increases at the rate at
which it removes Ca 2+ from the protein, allowing for a direct determination of
Ca 2+ off-rates. These techniques allow a rapid characterization of the Ca 2+ binding
and exchange properties of any Ca 2+ -binding protein. This information
allows for a more accurate prediction of a protein’s activation/inactivation profile
in response to cellular Ca 2+ transients.
2. Materials
2.1. Fluorescence Methods for Determining Ca2+ Affinity
1. A scanning fluorescence spectrophotometer.
2. 1 cm path length, four sides polished, 1 mL quartz cuvets.
3. A purified stock of Ca2+ -binding protein (typically 100–300 µM concentration).
4. A calibrated stock of CaCl2 (generally 0.5 M) and a calibrated (see Note 4) buffer
composed of 200 mM MOPS, 90 mM KCl, and 2 mM EGTA at pH 7.0.
5. A Ca2+ -titration computer printout (as generated by the Robinson and Potter [9],
Fabiato [10], or Schoenmaker et al. [11] computer programs) that shows the number
of microliters of your Ca2+ stock, which should be added to a particular volume
of your protein + buffer solution to obtain specific pCas.
6. Fluorescent Ca2+ indicators: Quin-2, Fura-2, or Fluo-3.
2.2. Methods of Monitoring Ca2+ Dissociation and Association
Rates from Proteins
1. A fluorescence stopped-flow spectrophotometer with rapid mixing kinetics and a
computer for data acquisition and analysis.
2. Purified Ca2+ -binding proteins, Quin-2, EGTA, and chelex resin.
3. Methods
3.1. Fluorescence Methods of Monitoring Ca2+ Binding
to Proteins
Fig. 1 shows a Ca2+ titration of F19W calmodulin as an example of a Ca2+ titration of a fluorescent Ca2+ -binding protein. Ca2+ binds half-maximally at
pCa 5.4 (4 × 10 –6 M) and produces a threefold increase in TRP fluorescence