into a three-state binding mechanism (Schemes 2 or 3). Similarly,
high salt concentrations may be used to show that electrostatic
interactions are important for the association of the proteins (Subheading 3.6.5).
3 Methods
3.1 Designing
and Performing
Binding Experiments
Experiments must be optimized with regard to sample availability,
protein concentrations, solubility, affinity, presumed mechanism,
etc. Other factors may also be important but these may only
become evident following initial pilot experiments. The result of a
kinetic experiment is a trace (or transient) in which fluorescence
(or absorbance, fluorescence polarization, etc.) changes over time.
In a typical binding experiment, if the fluorescent probe such as a
Trp is in protein A, then it is mixed rapidly with varying concentrations of an excess of protein B. However, it should not matter
whether protein A or B is in excess, the result should be the same
in either case (see Note 3).
1. Stopped-flow instruments use conventional arc lamp light
sources. Switch on the lamp 15–30 min before use such that
the light output is stable when starting the experiments.
2. It is important to perform kinetic experiments at a well-defined
temperature. Switch on the thermostat of your system (usually
a water bath), set the experimental temperature, and let the
system equilibrate before making any measurements. Use the
internal temperature probe as the experimental temperature
rather than the temperature of the water bath as the instrumental probe is closer to the mixing cell. Even if this instrumental
probe is not accurately reporting an exact temperature at the
point of measurement, it still allows experimental temperature
to be precisely reproduced. Depending on the magnitude of
the observed rate constants of the reaction under investigation,
the temperature should be set such that reliable data can be
recorded (i.e., rate constants within the stopped-flow range).
As a rule of thumb, rate constants increase by a factor of 2 for
every 10
C increase and measuring k obs at different temperatures is therefore a good control experiment to rule out artifacts (see Note 4). On the other hand, decreasing temperature
reduces collisional quenching from solvent and therefore
enhances fluorescence levels and the size of associated changes
in fluorescence during binding. Stopped-flow instruments can
even work at temperatures close to 0
C providing care is taken
that the system does not leak.
3. For fluorescence measurements, select an excitation wavelength and choose an appropriate fluorescence emission filter.
For example, Trp is typically excited around 280 nm and
Kinetics of IDP Binding
111
high salt concentrations may be used to show that electrostatic
interactions are important for the association of the proteins (Subheading 3.6.5).
3 Methods
3.1 Designing
and Performing
Binding Experiments
Experiments must be optimized with regard to sample availability,
protein concentrations, solubility, affinity, presumed mechanism,
etc. Other factors may also be important but these may only
become evident following initial pilot experiments. The result of a
kinetic experiment is a trace (or transient) in which fluorescence
(or absorbance, fluorescence polarization, etc.) changes over time.
In a typical binding experiment, if the fluorescent probe such as a
Trp is in protein A, then it is mixed rapidly with varying concentrations of an excess of protein B. However, it should not matter
whether protein A or B is in excess, the result should be the same
in either case (see Note 3).
1. Stopped-flow instruments use conventional arc lamp light
sources. Switch on the lamp 15–30 min before use such that
the light output is stable when starting the experiments.
2. It is important to perform kinetic experiments at a well-defined
temperature. Switch on the thermostat of your system (usually
a water bath), set the experimental temperature, and let the
system equilibrate before making any measurements. Use the
internal temperature probe as the experimental temperature
rather than the temperature of the water bath as the instrumental probe is closer to the mixing cell. Even if this instrumental
probe is not accurately reporting an exact temperature at the
point of measurement, it still allows experimental temperature
to be precisely reproduced. Depending on the magnitude of
the observed rate constants of the reaction under investigation,
the temperature should be set such that reliable data can be
recorded (i.e., rate constants within the stopped-flow range).
As a rule of thumb, rate constants increase by a factor of 2 for
every 10
C increase and measuring k obs at different temperatures is therefore a good control experiment to rule out artifacts (see Note 4). On the other hand, decreasing temperature
reduces collisional quenching from solvent and therefore
enhances fluorescence levels and the size of associated changes
in fluorescence during binding. Stopped-flow instruments can
even work at temperatures close to 0
C providing care is taken
that the system does not leak.
3. For fluorescence measurements, select an excitation wavelength and choose an appropriate fluorescence emission filter.
For example, Trp is typically excited around 280 nm and
Kinetics of IDP Binding
111