Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

384 Török et al.
actions in the cell. When interpreting the fluorescent signals, it has to be considered
that both the concentration and the liganded state of calmodulin may
change simultaneously. Vertebrate calmodulins can be modified most readily
on lysine residues. Several nonfluorescent lysine reagents have been attached
to calmodulin and, in most cases, the reagents predominantly modified Lys 75
(2). The higher reactivity of Lys 75 as opposed to the other lysine residues in the
protein, originates in its lower pK a value as determined by NMR spectroscopy
(3). Here, we have used the isomeric fluorescein dichlorotriazine, 5-DTAF,
which readily modifies amines in proteins. In this chapter, we use the example
of Lys 75-labeled FL-calmodulin, which has already been used for imaging in
living cells. We intend to provide a comprehensive approach including the synthesis
and analysis of the fluorescent calmodulin and its application into living
cells. We close with demonstrating how the activation state of calmodulin can
be monitored in living cells by confocal imaging.
2. Materials
2.1. Synthesis of FL-Calmodulin
1. 4 mM 5-DTAF (D-16, Molecular Probes), stock solution dissolved in dimethyl
formamide (DMF).
2. 60 µM pig brain calmodulin (4) in 200 mM Tris-HCl (pH 8.5) containing 20 mM
CaCl2. 3. PD10 Columns (Pharmacia Biotech).
4. Solvent A: 0.1% solution trifluoroacetic acid (TFA) (HPLC grade)/H2O. 5. Solvent B: 0.082% TFA solution/acetonitrile (HPLC grade).
6. Semipreparative Vydac C18 reverse-phase column 10 × 250 mm (Hichrom Ltd., UK).
7. 1 M Tris-HCl buffer, pH 7.5.
2.2. Characterization of Lys 75 Singly Labeled FL-Calmodulin
1. Freeze dried, desalted, singly labeled Lys 75 FL-calmodulin
2. Digestion mixture containing 100 mM NH 4HCO 3, pH 9.0, 2 mM EGTA, and
10 µg/mL trypsin, TPCK treated (Sigma, Aldrich, UK).
3. Solvent A: 0.1% solution trifluoroacetic acid (TFA) (HPLC grade)/H 2O.
4. Solvent B: 0.082% TFA solution/acetonitrile (HPLC grade).
5. Semipreparative Vydac C18 reverse-phase column 10 × 250 mm (Hichrom Ltd.).
6. Electrospray mass spectrometer.
2.3. Other NH 2-Reactive Fluorescent Probes
1. Dansyl chloride.
2. TA-Cl [2,4-dichloro-6-(4-N,N-diethylaminophenyl)-1,3,5-triazine].
3. Texas Red.
4. Cy5 [N-(N-hydoxy-succinimidyl-carboxypentyl)-N 1 -(ethyl)-indodicarbocyamine-5,5
1 -disulforate].