2. Site-directed mutagenesis is a powerful method to help establishing mechanisms since it can pinpoint the significance of
specific amino acid residues. In such cases, if the mutation is
made in the non-varied protein, it is good practice to use the
same stock solution for the varied species. Then, even if the
absolute concentration (and thus k on ) is not correct, any error
will cancel out when the effect of the mutation on free energy is
assessed when comparing mutant and wild type: ΔΔG ¼ RT ln
(k on
wild-type /k on
mutant ).
3. The fluorescence signal is adjusted with a voltage for the PMT.
If the varied protein is fluorescent at the wavelength of detection then the overall signal will increase linearly as protein
concentration is increased. There is then a risk that the detector
output becomes saturated requiring the PMT voltage to be
lowered. While the PMT voltage used during the experiment
does not affect the k obs values, it will change the fluorescence
end points and amplitudes of the kinetic traces. Thus, whenever
an amplitude analysis is included in a study, it is important to
set the voltage such that the whole range of protein concentrations can be measured at a single PMT voltage. However, a
PMT voltage of >300 V is desirable as below this the detector
output can become nonlinear.
4. The rate constant for folding, k F , is often not particularly
sensitive to changes in temperature. However, unless k F is
measured directly, for example, using ligand trapping of the
folded state [32], k obs for folding (¼k F + k U ) will be temperature dependent due to the contribution of k U even when
measured in an experiment where the experiment (and signal
change) monitors the folding reaction.
5. If nothing is known regarding the kinetics of a binding reaction, it is advisable to first perform equilibrium binding experiments with an appropriate technique to obtain the K d and also
to record fluorescence emission spectra of the respective protein(s) alone and in complex to help selection of an excitation
wavelength and detection emission filter. If the K d is in the
single μM range or tighter, then excite 1 μM protein A at
280 nm and record fluorescence emission between 310 and
450 nm on a fluorimeter. Then record emission for 5 μM of
protein B, if it contains any aromatic residues. Finally, take a
spectrum of the complex 1 μM A and 5 μM B, and calculate the
difference spectrum (AB À (A + B)). The difference spectrum
will show at which wavelengths the fluorescence will change
maximally during binding and assist selection of emission filter
as well as indicating the relative increase or decrease in signal
compared to the initial levels. Other affinities or fluorescent
128
Elin Karlsson and Per Jemth
specific amino acid residues. In such cases, if the mutation is
made in the non-varied protein, it is good practice to use the
same stock solution for the varied species. Then, even if the
absolute concentration (and thus k on ) is not correct, any error
will cancel out when the effect of the mutation on free energy is
assessed when comparing mutant and wild type: ΔΔG ¼ RT ln
(k on
wild-type /k on
mutant ).
3. The fluorescence signal is adjusted with a voltage for the PMT.
If the varied protein is fluorescent at the wavelength of detection then the overall signal will increase linearly as protein
concentration is increased. There is then a risk that the detector
output becomes saturated requiring the PMT voltage to be
lowered. While the PMT voltage used during the experiment
does not affect the k obs values, it will change the fluorescence
end points and amplitudes of the kinetic traces. Thus, whenever
an amplitude analysis is included in a study, it is important to
set the voltage such that the whole range of protein concentrations can be measured at a single PMT voltage. However, a
PMT voltage of >300 V is desirable as below this the detector
output can become nonlinear.
4. The rate constant for folding, k F , is often not particularly
sensitive to changes in temperature. However, unless k F is
measured directly, for example, using ligand trapping of the
folded state [32], k obs for folding (¼k F + k U ) will be temperature dependent due to the contribution of k U even when
measured in an experiment where the experiment (and signal
change) monitors the folding reaction.
5. If nothing is known regarding the kinetics of a binding reaction, it is advisable to first perform equilibrium binding experiments with an appropriate technique to obtain the K d and also
to record fluorescence emission spectra of the respective protein(s) alone and in complex to help selection of an excitation
wavelength and detection emission filter. If the K d is in the
single μM range or tighter, then excite 1 μM protein A at
280 nm and record fluorescence emission between 310 and
450 nm on a fluorimeter. Then record emission for 5 μM of
protein B, if it contains any aromatic residues. Finally, take a
spectrum of the complex 1 μM A and 5 μM B, and calculate the
difference spectrum (AB À (A + B)). The difference spectrum
will show at which wavelengths the fluorescence will change
maximally during binding and assist selection of emission filter
as well as indicating the relative increase or decrease in signal
compared to the initial levels. Other affinities or fluorescent
128
Elin Karlsson and Per Jemth