2 Materials
2.1 The
Stopped-Flow
Instrument
In the current chapter, we will mainly use stopped-flow experiments
as examples. However, the approach is valid for any kinetic method.
Whichever is the method of choice, the important thing is to have
control over potential artifacts and other caveats and to know the
limitations of the technique. Stopped flow and the other classical
kinetic methods perturb the equilibrium of the system and measure
the “relaxation” to the new equilibrium. In this respect, stopped
flow is very efficient since it mixes two solutions, which enables
large perturbations. The mixing time (“dead time”) of the very best
stopped-flow devices with T-type mixers is claimed to be around
1 ms; however, in our experience 2 ms is more common. The
mixing time is also dependent on the properties of the solvent
and the ratio of mixing (usually 1:1 in binding experiments but
for folding experiments often an asymmetric 10:1 mixing). Each
experimenter should determine the mixing time for their particular
instrument and setup using established model reactions (see Note
1). The mixing time and the kinetic amplitude determine how large
k obs values can be determined reliably in a particular experiment.
The observed kinetic amplitudes will usually start to decrease when
the reaction is too fast, i.e., when the time constant approaches the
mixing time. The instrument then captures only part of the kinetic
transient. For example, if the instrumental mixing time is 2 ms, an
average lifetime τ of the reaction in the same order would correspond to a k obs of 500 s
À1 . Then, after 2 ms 63% of the reaction is
complete, and the final 37% of the transient can be used for curve
fitting. This might be feasible if the signal-to-noise of the kinetic
amplitude is good enough. However, in practice, 200–400 s
À1 can
be the upper limit for measured k obs values. At the other end, slow
transients and long measuring times suffer from backflow of solutions in the tubings, photobleaching of the samples, or other
instrumental problems, which limit the measuring time to
10–100 s (k obs of 0.01–0.1 s
À1 ) depending on experimental setup
and protein samples. The temporal limitation of the instrument and
experiment must be judged on a case-by-case basis.
2.2 Protein Samples
In protein binding as well as folding studies using stopped flow,
fluorescence is the most common way to monitor the binding
and/or folding reaction. The great advantage with fluorescence is
that it is very sensitive to conformational changes. Any interactions
involving change of local environment of a Trp (or to some extent
Tyr) side chain, or other extrinsic fluorescent probes if such are
used, will translate into a change in fluorescence that can be monitored over time. However, one problem can be that there is no
native Trp or Tyr in the interacting proteins, or they are located in
positions not affected by the interaction. In such cases, a Trp could
Kinetics of IDP Binding
109
2.1 The
Stopped-Flow
Instrument
In the current chapter, we will mainly use stopped-flow experiments
as examples. However, the approach is valid for any kinetic method.
Whichever is the method of choice, the important thing is to have
control over potential artifacts and other caveats and to know the
limitations of the technique. Stopped flow and the other classical
kinetic methods perturb the equilibrium of the system and measure
the “relaxation” to the new equilibrium. In this respect, stopped
flow is very efficient since it mixes two solutions, which enables
large perturbations. The mixing time (“dead time”) of the very best
stopped-flow devices with T-type mixers is claimed to be around
1 ms; however, in our experience 2 ms is more common. The
mixing time is also dependent on the properties of the solvent
and the ratio of mixing (usually 1:1 in binding experiments but
for folding experiments often an asymmetric 10:1 mixing). Each
experimenter should determine the mixing time for their particular
instrument and setup using established model reactions (see Note
1). The mixing time and the kinetic amplitude determine how large
k obs values can be determined reliably in a particular experiment.
The observed kinetic amplitudes will usually start to decrease when
the reaction is too fast, i.e., when the time constant approaches the
mixing time. The instrument then captures only part of the kinetic
transient. For example, if the instrumental mixing time is 2 ms, an
average lifetime τ of the reaction in the same order would correspond to a k obs of 500 s
À1 . Then, after 2 ms 63% of the reaction is
complete, and the final 37% of the transient can be used for curve
fitting. This might be feasible if the signal-to-noise of the kinetic
amplitude is good enough. However, in practice, 200–400 s
À1 can
be the upper limit for measured k obs values. At the other end, slow
transients and long measuring times suffer from backflow of solutions in the tubings, photobleaching of the samples, or other
instrumental problems, which limit the measuring time to
10–100 s (k obs of 0.01–0.1 s
À1 ) depending on experimental setup
and protein samples. The temporal limitation of the instrument and
experiment must be judged on a case-by-case basis.
2.2 Protein Samples
In protein binding as well as folding studies using stopped flow,
fluorescence is the most common way to monitor the binding
and/or folding reaction. The great advantage with fluorescence is
that it is very sensitive to conformational changes. Any interactions
involving change of local environment of a Trp (or to some extent
Tyr) side chain, or other extrinsic fluorescent probes if such are
used, will translate into a change in fluorescence that can be monitored over time. However, one problem can be that there is no
native Trp or Tyr in the interacting proteins, or they are located in
positions not affected by the interaction. In such cases, a Trp could
Kinetics of IDP Binding
109