The slope of this plot reports on the extent of formation of the
mutated interaction(s) in the transition state. In the case of ACTR/
NCBD, the mutations were all deletion of hydrophobic moieties.
The slope of the plot was 0.18 suggesting that most native hydrophobic interactions have not formed in the conformation present at
the top of the transition state barrier. However, the scatter in the
data points suggests substantial local variation. These kinetic data
can be combined with molecular dynamics simulations to obtain a
clearer picture of the transition state. This combined approach
suggested that the transition state does indeed retain high levels
of disorder [28].
One recurring question in the IDP field is whether intrinsic
disorder per se promotes protein association as predicted by a “flycasting hypothesis” [29]. This is however not easy to test experimentally. Electrostatic interactions usually promote IDP interactions by increasing the association rate constant (Fig. 4b) masking
any effects of the intrinsic disorder. Therefore, the salt dependence
of several IDP interactions has been investigated to determine the
basal association rate constant in absence of electrostatic interactions (i.e., at extrapolated infinite ionic strength) [17, 21, 30,
31]. This parameter has been compared between different protein–protein interactions to assess the role of intrinsic disorder on
protein association [30]. While this might be the best experimental
way to address the fly-casting hypothesis, the conclusions so far are
not clear because of the limited number of experimental examples
in the literature.
Conclusions: Variation in buffer conditions and mutagenesis in
conjunction with detailed kinetic experiments provide clues to
binding mechanisms and the transition state of the interaction
for IDPs.
4 Notes
1. There is some confusion about what stopped-flow “dead time”
represents and we prefer the use of the term “mixing time”
which is the time from the true “time zero” of mixing (which
may be in negative time relative to the triggering of data
acquisition) and the first reliable data points in the kinetic traces
recorded. Time zero and mixing time can be determined in the
same simple experiment using a chemical reaction that is two
state and gives clean single exponential kinetics with good
signal to noise, for example, quenching of fluorescence of Nacetyl tryptophanamide (NATA) using N-bromosuccinimide
126
Elin Karlsson and Per Jemth
mutated interaction(s) in the transition state. In the case of ACTR/
NCBD, the mutations were all deletion of hydrophobic moieties.
The slope of the plot was 0.18 suggesting that most native hydrophobic interactions have not formed in the conformation present at
the top of the transition state barrier. However, the scatter in the
data points suggests substantial local variation. These kinetic data
can be combined with molecular dynamics simulations to obtain a
clearer picture of the transition state. This combined approach
suggested that the transition state does indeed retain high levels
of disorder [28].
One recurring question in the IDP field is whether intrinsic
disorder per se promotes protein association as predicted by a “flycasting hypothesis” [29]. This is however not easy to test experimentally. Electrostatic interactions usually promote IDP interactions by increasing the association rate constant (Fig. 4b) masking
any effects of the intrinsic disorder. Therefore, the salt dependence
of several IDP interactions has been investigated to determine the
basal association rate constant in absence of electrostatic interactions (i.e., at extrapolated infinite ionic strength) [17, 21, 30,
31]. This parameter has been compared between different protein–protein interactions to assess the role of intrinsic disorder on
protein association [30]. While this might be the best experimental
way to address the fly-casting hypothesis, the conclusions so far are
not clear because of the limited number of experimental examples
in the literature.
Conclusions: Variation in buffer conditions and mutagenesis in
conjunction with detailed kinetic experiments provide clues to
binding mechanisms and the transition state of the interaction
for IDPs.
4 Notes
1. There is some confusion about what stopped-flow “dead time”
represents and we prefer the use of the term “mixing time”
which is the time from the true “time zero” of mixing (which
may be in negative time relative to the triggering of data
acquisition) and the first reliable data points in the kinetic traces
recorded. Time zero and mixing time can be determined in the
same simple experiment using a chemical reaction that is two
state and gives clean single exponential kinetics with good
signal to noise, for example, quenching of fluorescence of Nacetyl tryptophanamide (NATA) using N-bromosuccinimide
126
Elin Karlsson and Per Jemth