structure shows an extended dumbbell shape, with the N- and
C-terminal domains connected by a solvent-exposed α-helix [5]
that is flexible in solution [6]. This flexibility is crucial for the
binding and activation of target enzymes.
The binding of Ca
2+ to CaM induces conformational changes
in its N- and C-terminal domains, resulting in the exposure of
hydrophobic pockets that are important for target protein binding
[7]. The structural basis of the interaction of CaM with target
proteins has largely been investigated using short synthetic peptides
corresponding to the binding domains of the intact target proteins.
This chapter describes methods for determining kinetic constants
for the binding of these synthetic peptides and of the intact
proteins.
1.2 The Choice
of Techniques
There are various ways to monitor changes in concentration of
reactants, intermediates, and products after mixing, but the most
common way is to use changes in optical signals (generally either
absorbance or fluorescence) which often accompany reactions.
Although absorbance can sometimes be used, fluorescence is
often preferred because of its greater sensitivity, particularly in
monitoring conformational changes. Such methods are continuous
with good time resolution but they seldom permit the direct determination of the concentrations of individual species. Alternatively,
samples may be taken from the reaction volume, mixed with a
chemical quenching agent to stop the reaction, and their contents
assessed by techniques such as HPLC. These methods can directly
determine the concentrations of different species, but are discontinuous and have a limited time resolution.
In all rapid mixing, or “flow” techniques, the reactant solutions
are driven at high velocity into a special mixing chamber. The
mixing and subsequent passage to the point of observation take a
finite amount of time, so that the mixed solution already has a
certain “age” before it can be observed. The interval between the
start of the mixing and the earliest possible observation time is
called the instrument’s dead time.
In stopped-flow, the commonest flow technique, the mixed
solution rapidly flows into an observation chamber, where it is
stopped and monitored by recording the change in some suitable
optical signal as a function of time (see Notes 1 and 2). The dead
time of a stopped-flow instrument is typically 1–2 ms and reactions
occurring on a faster time scale cannot be studied.
In quenched-flow, the reactants are mixed and flow down an
“aging tube” at constant velocity before mixing with a “quenching
agent,” generally acid, that stops the reaction. The quenched reaction mixture is then analyzed using an appropriate method, such as
HPLC. Because the age of the quenched sample is determined by
the flow rate and the flow tube volume, a series of time points is
built up by doing experiments with different flow rates and/or tube
84
Stephen R. Martin and Maria J. Schilstra
C-terminal domains connected by a solvent-exposed α-helix [5]
that is flexible in solution [6]. This flexibility is crucial for the
binding and activation of target enzymes.
The binding of Ca
2+ to CaM induces conformational changes
in its N- and C-terminal domains, resulting in the exposure of
hydrophobic pockets that are important for target protein binding
[7]. The structural basis of the interaction of CaM with target
proteins has largely been investigated using short synthetic peptides
corresponding to the binding domains of the intact target proteins.
This chapter describes methods for determining kinetic constants
for the binding of these synthetic peptides and of the intact
proteins.
1.2 The Choice
of Techniques
There are various ways to monitor changes in concentration of
reactants, intermediates, and products after mixing, but the most
common way is to use changes in optical signals (generally either
absorbance or fluorescence) which often accompany reactions.
Although absorbance can sometimes be used, fluorescence is
often preferred because of its greater sensitivity, particularly in
monitoring conformational changes. Such methods are continuous
with good time resolution but they seldom permit the direct determination of the concentrations of individual species. Alternatively,
samples may be taken from the reaction volume, mixed with a
chemical quenching agent to stop the reaction, and their contents
assessed by techniques such as HPLC. These methods can directly
determine the concentrations of different species, but are discontinuous and have a limited time resolution.
In all rapid mixing, or “flow” techniques, the reactant solutions
are driven at high velocity into a special mixing chamber. The
mixing and subsequent passage to the point of observation take a
finite amount of time, so that the mixed solution already has a
certain “age” before it can be observed. The interval between the
start of the mixing and the earliest possible observation time is
called the instrument’s dead time.
In stopped-flow, the commonest flow technique, the mixed
solution rapidly flows into an observation chamber, where it is
stopped and monitored by recording the change in some suitable
optical signal as a function of time (see Notes 1 and 2). The dead
time of a stopped-flow instrument is typically 1–2 ms and reactions
occurring on a faster time scale cannot be studied.
In quenched-flow, the reactants are mixed and flow down an
“aging tube” at constant velocity before mixing with a “quenching
agent,” generally acid, that stops the reaction. The quenched reaction mixture is then analyzed using an appropriate method, such as
HPLC. Because the age of the quenched sample is determined by
the flow rate and the flow tube volume, a series of time points is
built up by doing experiments with different flow rates and/or tube
84
Stephen R. Martin and Maria J. Schilstra