2 Materials
2.1 Site-Directed
Mutagenesis
Site mutations is critical in understanding protein folding and in
addressing the structural features of transiently accumulated state.
Consequently, these issues have been previously discussed extensively [2, 13]. In brief, we here recall how the introduction of
nonnative interactions and steric hindrance should be avoided, as
well as any destabilizing charge. The mutagenesis can be done with
Quick-Change Lightning Site-Directed Mutagenesis kit (Agilent
Technologies) according to the manufacturer’s instructions and all
mutations must be then be confirmed by DNA sequencing. If
needed, it is possible to perform side directed mutagenesis to
introduce a tryptophan as an optical probe for fluorescence monitoring experiments.
2.2 Equilibrium
Unfolding Experiments
Equilibrium unfolding experiments are performed on a standard
spectrofluorometer, equipped with two monochromators but in
the excitation and in the emission paths. Tryptophan containing
PDZ proteins are excited at 280 nm, at a constant concentration
ranging around the μM scale, and emission spectra are recorded
between 300 and 400 nm, at increasing denaturant concentration.
Experiments can be performed at varying temperatures, using a
quartz cuvette with a path length of 1 cm. The fluorescence emission is then analyzed quantitatively following a two-state model.
2.3 Folding Kinetic
Experiments
Rapid mixing for kinetics folding and unfolding experiments can be
carried out on a stopped-flow device with an excitation wavelength
of 280 nm. An appropriate cut-off glass filter must be employed to
measure fluorescence emission. The protein concentration should
be in the μM range and the denaturant concentration may typically
vary between 0 and 8 M. Degassing solutions may help and reduce
the presence of mixing bubbles during stopped-flow experiments.
3 Methods
Protein folding can be studied in vitro by subjecting the protein to
changing condition, with the goal to infer a progressive denaturation. The loss of native structure can be monitored by measuring
changes in absorbance or fluorescence as well as using circular
dichroism and NMR spectroscopy. Denaturants like guanidinium
or urea are most commonly used to study protein folding because
they disrupt a large number of weak interactions, which allows us to
study the progressive loss of structure by varying concentrations of
denaturant [14]. Denaturation can also be caused by a change of
temperature and pH [1]. In these cases, however, the dependence
of the unfolding free energy is more complex and, therefore, we will
not discuss it in this brief Methods.
150
Candice Gautier and Stefano Gianni
2.1 Site-Directed
Mutagenesis
Site mutations is critical in understanding protein folding and in
addressing the structural features of transiently accumulated state.
Consequently, these issues have been previously discussed extensively [2, 13]. In brief, we here recall how the introduction of
nonnative interactions and steric hindrance should be avoided, as
well as any destabilizing charge. The mutagenesis can be done with
Quick-Change Lightning Site-Directed Mutagenesis kit (Agilent
Technologies) according to the manufacturer’s instructions and all
mutations must be then be confirmed by DNA sequencing. If
needed, it is possible to perform side directed mutagenesis to
introduce a tryptophan as an optical probe for fluorescence monitoring experiments.
2.2 Equilibrium
Unfolding Experiments
Equilibrium unfolding experiments are performed on a standard
spectrofluorometer, equipped with two monochromators but in
the excitation and in the emission paths. Tryptophan containing
PDZ proteins are excited at 280 nm, at a constant concentration
ranging around the μM scale, and emission spectra are recorded
between 300 and 400 nm, at increasing denaturant concentration.
Experiments can be performed at varying temperatures, using a
quartz cuvette with a path length of 1 cm. The fluorescence emission is then analyzed quantitatively following a two-state model.
2.3 Folding Kinetic
Experiments
Rapid mixing for kinetics folding and unfolding experiments can be
carried out on a stopped-flow device with an excitation wavelength
of 280 nm. An appropriate cut-off glass filter must be employed to
measure fluorescence emission. The protein concentration should
be in the μM range and the denaturant concentration may typically
vary between 0 and 8 M. Degassing solutions may help and reduce
the presence of mixing bubbles during stopped-flow experiments.
3 Methods
Protein folding can be studied in vitro by subjecting the protein to
changing condition, with the goal to infer a progressive denaturation. The loss of native structure can be monitored by measuring
changes in absorbance or fluorescence as well as using circular
dichroism and NMR spectroscopy. Denaturants like guanidinium
or urea are most commonly used to study protein folding because
they disrupt a large number of weak interactions, which allows us to
study the progressive loss of structure by varying concentrations of
denaturant [14]. Denaturation can also be caused by a change of
temperature and pH [1]. In these cases, however, the dependence
of the unfolding free energy is more complex and, therefore, we will
not discuss it in this brief Methods.
150
Candice Gautier and Stefano Gianni