more stable the higher the concentration of the protein added in
excess. Despite this, it was possible to measure the coupled folding
and binding reaction between N TAIL and XD using an engineered
Trp residue as fluorescence probe. The experimental traces
obtained from the temperature jumps were well described by a
single exponential as in the first case study. However, in contrast
to p53TAD/MDM2, k obs values were not increasing linearly with
N TAIL concentration as expected from a one-step binding mechanism, but instead displayed a hyperbolic dependence with a limiting
value around 1000 s
À1
. Such behavior is consistent with a two-step
mechanism (three state). While a two-step mechanism should theoretically yield double exponential traces, it is quite common that
one of the kinetic phases is not detected because it is too fast for the
instrument or the amplitude is too small, or both. There has been
considerable interest in whether three-state mechanisms involving
IDPs follow an induced fit or conformational selection model.
Induced fit requires the two proteins associate first and then fold
into the equilibrium conformation of the complex (Scheme 2).
Conformational selection requires that folding of a fraction of the
IDP population precedes the binding step (Scheme 3). With regard
to IDP interactions, the question often oversimplifies the problem
since it is very hard to imagine a disordered polypeptide folding
into the precise geometry of the complex before associating with its
folded binding partner. However, in the case of the N TAIL binding
motif that is helical this might be possible since isolated helices may
form transiently in absence of tertiary structure. The authors used a
kinetic trick first devised by Olson et al. [22] and later rediscovered
[23] to demonstrate that the N TAIL /XD interaction indeed follows
induced fit. This requires changing the concentration of N TAIL and
XD in separate experiments. If k obs shows a hyperbolic dependence
in both cases, the binding occurs before the conformational change
(folding) with induced fit. If the dependence is hyperbolic for one
of the proteins but linear for the other, folding happens before
binding in conformational selection.
The authors could also demonstrate that the conformational
change associated with the hyperbolic behavior of k obs was associated with the folding of N TAIL using the helix-stabilizing agent,
2,2,2-trifluoroethanol (TFE). Addition of TFE produced increases
of k obs at high [N TAIL ] and a less pronounced hyperbolic behavior.
This is consistent with a lower barrier and larger rate constant for
folding of the N TAIL helix (an increase of k 2 in Scheme 2). Thus,
N TAIL binds to XD in a disordered state and folds into an α-helix in
a second step.
Conclusions: Single exponential binding kinetics where k obs increases
hyperbolically with ligand concentration is consistent with a two-step
(three state) binding mechanism. This could be an induced fit
Kinetics of IDP Binding
121
excess. Despite this, it was possible to measure the coupled folding
and binding reaction between N TAIL and XD using an engineered
Trp residue as fluorescence probe. The experimental traces
obtained from the temperature jumps were well described by a
single exponential as in the first case study. However, in contrast
to p53TAD/MDM2, k obs values were not increasing linearly with
N TAIL concentration as expected from a one-step binding mechanism, but instead displayed a hyperbolic dependence with a limiting
value around 1000 s
À1
. Such behavior is consistent with a two-step
mechanism (three state). While a two-step mechanism should theoretically yield double exponential traces, it is quite common that
one of the kinetic phases is not detected because it is too fast for the
instrument or the amplitude is too small, or both. There has been
considerable interest in whether three-state mechanisms involving
IDPs follow an induced fit or conformational selection model.
Induced fit requires the two proteins associate first and then fold
into the equilibrium conformation of the complex (Scheme 2).
Conformational selection requires that folding of a fraction of the
IDP population precedes the binding step (Scheme 3). With regard
to IDP interactions, the question often oversimplifies the problem
since it is very hard to imagine a disordered polypeptide folding
into the precise geometry of the complex before associating with its
folded binding partner. However, in the case of the N TAIL binding
motif that is helical this might be possible since isolated helices may
form transiently in absence of tertiary structure. The authors used a
kinetic trick first devised by Olson et al. [22] and later rediscovered
[23] to demonstrate that the N TAIL /XD interaction indeed follows
induced fit. This requires changing the concentration of N TAIL and
XD in separate experiments. If k obs shows a hyperbolic dependence
in both cases, the binding occurs before the conformational change
(folding) with induced fit. If the dependence is hyperbolic for one
of the proteins but linear for the other, folding happens before
binding in conformational selection.
The authors could also demonstrate that the conformational
change associated with the hyperbolic behavior of k obs was associated with the folding of N TAIL using the helix-stabilizing agent,
2,2,2-trifluoroethanol (TFE). Addition of TFE produced increases
of k obs at high [N TAIL ] and a less pronounced hyperbolic behavior.
This is consistent with a lower barrier and larger rate constant for
folding of the N TAIL helix (an increase of k 2 in Scheme 2). Thus,
N TAIL binds to XD in a disordered state and folds into an α-helix in
a second step.
Conclusions: Single exponential binding kinetics where k obs increases
hyperbolically with ligand concentration is consistent with a two-step
(three state) binding mechanism. This could be an induced fit
Kinetics of IDP Binding
121