3 Methods
3.1 Simple
Bimolecular Binding
Reactions
Many protein–ligand interactions are simple, reversible secondorder reactions (Scheme B), and we focus first on the determination of the rate constants for such processes. Stopped-flow kinetic
studies of reactions of this type are generally performed under
pseudo-first-order conditions with the concentration of one of
the reagents (either P or L) at least tenfold higher than the other
(see Subheading 1.3). The choice of the component to be used in
excess may be dictated by availability of material; if this is not the
case, it should be selected so that the ratio of signal change to total
background signal is maximized (see Note 6). In this section, we
describe an investigation of the interaction of Ca 4 –CaM with a
fluorescently labeled peptide NMp. A typical experiment will
involve the following steps:
1. Perform a few “blank” shots with the fluorescent component
alone to establish the starting fluorescence level.
2. Perform initial measurements with the component in excess,
[X tot ], at a tenfold excess. The actual concentration selected
will depend on the instrument being used and on the intensity
of the fluorophore but in the studies reported here the protein
is generally the component in excess and initial measurements
typically start with the protein concentration in the range
1–10 μM.
3. Optimize the concentration of [X tot ] if necessary; If the reaction is too fast (more or less complete within the dead time),
the concentrations of both components will need to be
reduced. The lowest usable value of [X tot ] will be that which
still maintains pseudo-first-order conditions but is high enough
to give good S/N. If the reaction is slow then the concentrations can be increased if this improves S/N.
4. When a suitable signal change has been obtained examine it
over a wide range of time scales in order to confirm that there is
only a single exponential process (see Fig. 2). Slow changes
sometimes observed in fluorescence measurements can be
caused by photobleaching of the fluorophore. This is not usually a problem on time scales of <1 s and can be quantified by
mixing the fluorophore with buffer and recording any decrease
in fluorescence intensity. Reducing the excitation slit width will
reduce photobleaching.
5. Perform measurements at several different values of
[X tot ]. Inspect all transients and average at least five at each
concentration for subsequent analysis. If inconsistent results
Calmodulin Target Interactions
89
3.1 Simple
Bimolecular Binding
Reactions
Many protein–ligand interactions are simple, reversible secondorder reactions (Scheme B), and we focus first on the determination of the rate constants for such processes. Stopped-flow kinetic
studies of reactions of this type are generally performed under
pseudo-first-order conditions with the concentration of one of
the reagents (either P or L) at least tenfold higher than the other
(see Subheading 1.3). The choice of the component to be used in
excess may be dictated by availability of material; if this is not the
case, it should be selected so that the ratio of signal change to total
background signal is maximized (see Note 6). In this section, we
describe an investigation of the interaction of Ca 4 –CaM with a
fluorescently labeled peptide NMp. A typical experiment will
involve the following steps:
1. Perform a few “blank” shots with the fluorescent component
alone to establish the starting fluorescence level.
2. Perform initial measurements with the component in excess,
[X tot ], at a tenfold excess. The actual concentration selected
will depend on the instrument being used and on the intensity
of the fluorophore but in the studies reported here the protein
is generally the component in excess and initial measurements
typically start with the protein concentration in the range
1–10 μM.
3. Optimize the concentration of [X tot ] if necessary; If the reaction is too fast (more or less complete within the dead time),
the concentrations of both components will need to be
reduced. The lowest usable value of [X tot ] will be that which
still maintains pseudo-first-order conditions but is high enough
to give good S/N. If the reaction is slow then the concentrations can be increased if this improves S/N.
4. When a suitable signal change has been obtained examine it
over a wide range of time scales in order to confirm that there is
only a single exponential process (see Fig. 2). Slow changes
sometimes observed in fluorescence measurements can be
caused by photobleaching of the fluorophore. This is not usually a problem on time scales of <1 s and can be quantified by
mixing the fluorophore with buffer and recording any decrease
in fluorescence intensity. Reducing the excitation slit width will
reduce photobleaching.
5. Perform measurements at several different values of
[X tot ]. Inspect all transients and average at least five at each
concentration for subsequent analysis. If inconsistent results
Calmodulin Target Interactions
89