be engineered at an appropriate position using site-directed mutagenesis. However, this might affect the kinetics and affinity of the
interaction and proper controls must be performed. For example,
k off for the wild-type protein variant should be determined in a
displacement reaction described below and the overall K d with
isothermal titration calorimetry [12] or other equilibrium method.
In this way, the K d , k off , and k on (¼k off /K d ) can be compared for
wild-type and Trp variant before extensive kinetic experiments are
initiated. Another control is to put the Trp at a different position or
engineer a fluorescence signal into the other binding protein and
check for consistency in the equilibrium and kinetic parameters. In
fact, a screen of different Trp variants is usually necessary to find the
most appropriate one for detailed kinetic studies.
In experiments involving coupled binding and folding of IDPs,
it is sometimes appropriate to use synthesized peptides
corresponding to only the disordered region involved in binding.
Peptide synthesis provides an easy route to introduce Trp into the
peptide at a suitable position. In addition, peptides can be chemically ligated to larger and often brighter fluorophores during synthesis. This has advantages in giving a discrete and selectable
wavelength for this component of the interaction. Chemical labeling of expressed proteins with these fluorophores is also done
routinely and has been used to study IDPs [13] but requires
additional steps.
In order to obtain an accurate k on value, the concentration of
the varied protein or peptide must be known accurately. Absorbance is by far the best method for this [12, 14] particularly if a Tyr,
Trp, or other extrinsic fluorophore with a strong absorbance in the
UV or visible spectrum is present. If this is not possible, as in
Subheading 3.6.3 below, then absorbance at 205 nm can be used
but the concentration should be confirmed using quantitative
amino acid analysis. The concentration is usually the main source
of error in k on and K d determinations and this should be kept in
mind when interpreting data (see Note 2).
2.3 Buffers
Buffers should be selected in which the proteins of interest remain
stable for the duration of stopped-flow measurement that may be
considerably longer than the time of individual kinetic runs. Systematic changes in buffer composition can also be used to investigate binding mechanism. For example, properties such as ionic
strength, pH, chaotropic, or antichaotropic activity have all been
classically used in protein folding studies and can likewise be applied
to IDP interactions [15–17]. Such variation in buffer conditions
can stabilize or destabilize intermediates, or affect the transition
state(s) of the binding reaction. If an intermediate is stabilized, it
may be detected in the kinetic experiment and in some cases be
shown to be present on the productive binding pathway. In other
words, an apparent two-state binding (Scheme 1) could be “tuned”
110
Elin Karlsson and Per Jemth
interaction and proper controls must be performed. For example,
k off for the wild-type protein variant should be determined in a
displacement reaction described below and the overall K d with
isothermal titration calorimetry [12] or other equilibrium method.
In this way, the K d , k off , and k on (¼k off /K d ) can be compared for
wild-type and Trp variant before extensive kinetic experiments are
initiated. Another control is to put the Trp at a different position or
engineer a fluorescence signal into the other binding protein and
check for consistency in the equilibrium and kinetic parameters. In
fact, a screen of different Trp variants is usually necessary to find the
most appropriate one for detailed kinetic studies.
In experiments involving coupled binding and folding of IDPs,
it is sometimes appropriate to use synthesized peptides
corresponding to only the disordered region involved in binding.
Peptide synthesis provides an easy route to introduce Trp into the
peptide at a suitable position. In addition, peptides can be chemically ligated to larger and often brighter fluorophores during synthesis. This has advantages in giving a discrete and selectable
wavelength for this component of the interaction. Chemical labeling of expressed proteins with these fluorophores is also done
routinely and has been used to study IDPs [13] but requires
additional steps.
In order to obtain an accurate k on value, the concentration of
the varied protein or peptide must be known accurately. Absorbance is by far the best method for this [12, 14] particularly if a Tyr,
Trp, or other extrinsic fluorophore with a strong absorbance in the
UV or visible spectrum is present. If this is not possible, as in
Subheading 3.6.3 below, then absorbance at 205 nm can be used
but the concentration should be confirmed using quantitative
amino acid analysis. The concentration is usually the main source
of error in k on and K d determinations and this should be kept in
mind when interpreting data (see Note 2).
2.3 Buffers
Buffers should be selected in which the proteins of interest remain
stable for the duration of stopped-flow measurement that may be
considerably longer than the time of individual kinetic runs. Systematic changes in buffer composition can also be used to investigate binding mechanism. For example, properties such as ionic
strength, pH, chaotropic, or antichaotropic activity have all been
classically used in protein folding studies and can likewise be applied
to IDP interactions [15–17]. Such variation in buffer conditions
can stabilize or destabilize intermediates, or affect the transition
state(s) of the binding reaction. If an intermediate is stabilized, it
may be detected in the kinetic experiment and in some cases be
shown to be present on the productive binding pathway. In other
words, an apparent two-state binding (Scheme 1) could be “tuned”
110
Elin Karlsson and Per Jemth