consistent with a two-step binding mechanism (three state) (see
Note 15). Curve fitting using Eqs. 4 and 5 can usually not distinguish induced fit from conformational selection when the conformational change is fast. However, if fluorescence yields can be assigned to
free proteins, intermediate, and bound complex, the kinetic amplitudes can be used to rule out mechanisms. If experimentally possible,
the concentrations of both proteins should be varied to assess the
hyperbolic dependence of the slow kinetic phase.
3.6.4 Case Study 4: HPV
E7 and Rb, a Multistep
Binding with Several
Kinetic Phases
An example of a multistep binding mechanism deduced by
stopped-flow kinetics is that for the intrinsically disordered
N-terminus of the human papillomavirus E7 protein (HPV E7)
and the folded human protein, Retinoblastoma tumor suppressor
(Rb) [13]. The interaction acts to inhibit the Rb protein, thus
promoting cell proliferation and HPV virus replication. In this
work, a FITC label on HPV E7 was used as fluorescence probe
giving the advantage that low concentrations of labeled HPV E7
protein could be used (5–50 nM). The authors compared the
binding kinetics of HPV E7 as a short peptide containing only
the binding motif with the kinetics of the entire disordered
N-terminus. The shorter peptide displayed two-state kinetics
while the longer disordered region followed a four-state mechanism. Interestingly, the longer protein bound 10 times tighter to
Rb, illustrating the important role of disordered regions outside
the designated interacting binding motif. Using experiments at a
range of NaCl concentrations, the authors also highlighted the
role of electrostatics in this IDP interaction and how they can be
characterized. Following extensive kinetic experiments at different [NaCl] and with structural considerations, the authors concluded that the four-state mechanism most likely involve initial
conformational selection in the disordered HPV E7 protein
(about 50% is in a binding competent conformation in the free
state) which is followed by association to Rb and a rearrangement of the complex. While a detailed description of the work is
beyond the scope of this protocol, it is recommended as further
reading illustrating an approach to very complex binding
kinetics.
Conclusions: Triple exponential binding kinetics proves that the
interaction involves (at least) three distinct events (four state). If
one kinetic phase increases linearly with [B] and the other two appears
constant or hyperbolic, the experiment is consistent with one association step and two conformational changes. If one kinetic phase
decreases with [B], it suggests that the initial step is a slow conformational change in A or B.
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Elin Karlsson and Per Jemth
Note 15). Curve fitting using Eqs. 4 and 5 can usually not distinguish induced fit from conformational selection when the conformational change is fast. However, if fluorescence yields can be assigned to
free proteins, intermediate, and bound complex, the kinetic amplitudes can be used to rule out mechanisms. If experimentally possible,
the concentrations of both proteins should be varied to assess the
hyperbolic dependence of the slow kinetic phase.
3.6.4 Case Study 4: HPV
E7 and Rb, a Multistep
Binding with Several
Kinetic Phases
An example of a multistep binding mechanism deduced by
stopped-flow kinetics is that for the intrinsically disordered
N-terminus of the human papillomavirus E7 protein (HPV E7)
and the folded human protein, Retinoblastoma tumor suppressor
(Rb) [13]. The interaction acts to inhibit the Rb protein, thus
promoting cell proliferation and HPV virus replication. In this
work, a FITC label on HPV E7 was used as fluorescence probe
giving the advantage that low concentrations of labeled HPV E7
protein could be used (5–50 nM). The authors compared the
binding kinetics of HPV E7 as a short peptide containing only
the binding motif with the kinetics of the entire disordered
N-terminus. The shorter peptide displayed two-state kinetics
while the longer disordered region followed a four-state mechanism. Interestingly, the longer protein bound 10 times tighter to
Rb, illustrating the important role of disordered regions outside
the designated interacting binding motif. Using experiments at a
range of NaCl concentrations, the authors also highlighted the
role of electrostatics in this IDP interaction and how they can be
characterized. Following extensive kinetic experiments at different [NaCl] and with structural considerations, the authors concluded that the four-state mechanism most likely involve initial
conformational selection in the disordered HPV E7 protein
(about 50% is in a binding competent conformation in the free
state) which is followed by association to Rb and a rearrangement of the complex. While a detailed description of the work is
beyond the scope of this protocol, it is recommended as further
reading illustrating an approach to very complex binding
kinetics.
Conclusions: Triple exponential binding kinetics proves that the
interaction involves (at least) three distinct events (four state). If
one kinetic phase increases linearly with [B] and the other two appears
constant or hyperbolic, the experiment is consistent with one association step and two conformational changes. If one kinetic phase
decreases with [B], it suggests that the initial step is a slow conformational change in A or B.
124
Elin Karlsson and Per Jemth