related to the displacement. The latter phase is avoided if
[B] ) K d , but a higher concentration of C is then necessary
to make the AB dissociation irreversible. To reduce the concentration of free A, it could be useful to have a small excess of
B over A, e.g., 1 μM A and 2 μM B (resulting in 59% AB
complex if K d ¼ 1 μM).
13. An attractive feature of displacement experiment is that any
errors in the concentrations of A, B, or C do not matter, since
there is no concentration-dependence of k obs at high enough
concentration of C. Measurement of the overall dissociation
rate constant determined in a displacement experiment at this
limit is therefore one of the more accurate kinetic parameters
that can be obtained.
14. Fast conformational changes occurring before binding are difficult to distinguish from an induced fit model. However, when
a slow conformational change precedes binding, a very characteristic decrease of k obs occurs upon increasing protein/ligand
concentration. In such cases, it is straightforward to rule out
induced fit and favor conformational selection. Experimentally,
this will appear exactly as a displacement experiment where the
dissociation of the protein–protein complex corresponds to the
intramolecular conformational change. Considering Scheme
3, in the rare perfectly intermediate case between “fast” and
“slow” pre-equilibrium where k 1 ¼ k À1 ¼ k 2 , the slow phase
will have a constant value (the same as the microscopic rate
constants) at all concentrations of B.
15. A triangular mechanism, in which binding can occur via either
an intermediate or directly to the bound complex, will also give
double exponential kinetics since it is still a three-state mechanism where the third step is not independent of the other two.
Likewise, a square mechanism, which is obtained if Schemes
2 and 3 are combined, will theoretically give triple exponential
kinetics.
References
1. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16:18–29.
https://doi.org/10.1038/nrm3920
2. Fuxreiter M (2019) Fold or not to fold upon
binding - does it really matter? Curr Opin
Struct Biol 54:19
3. Ivarsson Y, Jemth P (2018) Affinity and specificity of motif-based protein-protein interactions. Curr Opin Struct Biol 54:26–33.
https://doi.org/10.1016/j.sbi.2018.09.009
4. Dogan J, Gianni S, Jemth P (2014) The binding mechanisms of intrinsically disordered
proteins. Phys Chem Chem Phys 16:6323.
https://doi.org/10.1039/c3cp54226b
5. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an
intrinsically disordered protein. Nature
447:1021–1025. https://doi.org/10.1038/
nature05858
6. Dogan J, Jemth P (2014) Only kinetics can
prove conformational selection. Biophys J
107:1997–1998. https://doi.org/10.1016/j.
bpj.2014.08.037
7. Schneider R, Blackledge M, Jensen MR (2018)
Elucidating binding mechanisms and dynamics
Kinetics of IDP Binding
131
[B] ) K d , but a higher concentration of C is then necessary
to make the AB dissociation irreversible. To reduce the concentration of free A, it could be useful to have a small excess of
B over A, e.g., 1 μM A and 2 μM B (resulting in 59% AB
complex if K d ¼ 1 μM).
13. An attractive feature of displacement experiment is that any
errors in the concentrations of A, B, or C do not matter, since
there is no concentration-dependence of k obs at high enough
concentration of C. Measurement of the overall dissociation
rate constant determined in a displacement experiment at this
limit is therefore one of the more accurate kinetic parameters
that can be obtained.
14. Fast conformational changes occurring before binding are difficult to distinguish from an induced fit model. However, when
a slow conformational change precedes binding, a very characteristic decrease of k obs occurs upon increasing protein/ligand
concentration. In such cases, it is straightforward to rule out
induced fit and favor conformational selection. Experimentally,
this will appear exactly as a displacement experiment where the
dissociation of the protein–protein complex corresponds to the
intramolecular conformational change. Considering Scheme
3, in the rare perfectly intermediate case between “fast” and
“slow” pre-equilibrium where k 1 ¼ k À1 ¼ k 2 , the slow phase
will have a constant value (the same as the microscopic rate
constants) at all concentrations of B.
15. A triangular mechanism, in which binding can occur via either
an intermediate or directly to the bound complex, will also give
double exponential kinetics since it is still a three-state mechanism where the third step is not independent of the other two.
Likewise, a square mechanism, which is obtained if Schemes
2 and 3 are combined, will theoretically give triple exponential
kinetics.
References
1. Wright PE, Dyson HJ (2015) Intrinsically disordered proteins in cellular signalling and regulation. Nat Rev Mol Cell Biol 16:18–29.
https://doi.org/10.1038/nrm3920
2. Fuxreiter M (2019) Fold or not to fold upon
binding - does it really matter? Curr Opin
Struct Biol 54:19
3. Ivarsson Y, Jemth P (2018) Affinity and specificity of motif-based protein-protein interactions. Curr Opin Struct Biol 54:26–33.
https://doi.org/10.1016/j.sbi.2018.09.009
4. Dogan J, Gianni S, Jemth P (2014) The binding mechanisms of intrinsically disordered
proteins. Phys Chem Chem Phys 16:6323.
https://doi.org/10.1039/c3cp54226b
5. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an
intrinsically disordered protein. Nature
447:1021–1025. https://doi.org/10.1038/
nature05858
6. Dogan J, Jemth P (2014) Only kinetics can
prove conformational selection. Biophys J
107:1997–1998. https://doi.org/10.1016/j.
bpj.2014.08.037
7. Schneider R, Blackledge M, Jensen MR (2018)
Elucidating binding mechanisms and dynamics
Kinetics of IDP Binding
131