protein, in our case a PDZ domain. As an example, some proteins
may have a low Tm associated with a large ΔC p which will confer a
great stability in vivo. In parallel, PDZs with a mass > 5000 Daltons, such as proteins, form well-defined structures that undergo
thermally induced conformational changes [34]. These structural
rearrangements result in the absorption of heat caused by the
redistribution of noncovalent bonds. Differential scanning calorimeters measure this heat uptake.
Concerning NanoDSF, this technique records the intrinsic
fluorescence of Tryptophan (Trp) and Tyrosine (Tyr) residues,
which are very sensitive to changes in their local environment.
Thermal unfolding is measured by monitoring the intrinsic Trp
and Tyr fluorescence intensity, and the position of the emission
maximum as a function of temperature. The fluorescence intensity
ratio between 330 and 350 nm is defined as an empirical parameter
to monitor the evolution of the microenvironment of the aromatic
residues during protein denaturation throughout the temperature
increases. This ratio sharply increases/decreases during thermal
unfolding, allowing to determine a Tm value [35]. The applicability
of nanoDSF is highly dependent on the presence of Trp and Tyr in
the folded core of the PDZ that are exposed upon unfolding.
Moreover, it is necessary to exclude that the observed signal
changes are caused by aggregation, as this will also lead to variations
in the fluorophore environment. A back-scattering measurement
can also be performed to determine if aggregation occurs (before or
concomitantly with the denaturation). The characteristics of the
temperature gradient are essential as it is related to the activation
energy via the Arrhenius equation [6]. Typically, a heating rate of
1
C/min is applied. Overall, comparing the melting temperatures
(ΔT m ) in different buffer compositions allows researchers to define
the optimal buffer condition as an increase in Tm corresponds to a
better thermal stability and to a reduced conformational flexibility.
1.4.3 Nuclear Magnetic
Resonance (NMR)
NMR is based on the measure of the absorption of radiofrequency
(RF) radiation by an atomic nucleus located in a strong magnetic
field. The principle of NMR is that atomic nuclei, with an odd
number of protons (
1 H,
13
C,
15
N,
31
P, . . .), neutrons, or both,
have an intrinsic nuclear spin. When an atomic nucleus with a
nonzero spin is placed in a magnetic field, the nuclear spin aligned
in the same direction or in the opposite direction to the field.
Different energies characterize these two types of nuclear spin
alignment, and the application of a magnetic field facilitates the
degeneration of nuclear spins. An atomic nucleus whose spin is
aligned with the field will have less energy than when its spin is
aligned in the opposite direction of the field.
The energy of an NMR transition depends on the magnetic
field strength as well as on the proportionality factor applied to each
nucleus called the gyromagnetic ratio. The local environment
100
Ce ´ lia Caillet-Saguy et al.
may have a low Tm associated with a large ΔC p which will confer a
great stability in vivo. In parallel, PDZs with a mass > 5000 Daltons, such as proteins, form well-defined structures that undergo
thermally induced conformational changes [34]. These structural
rearrangements result in the absorption of heat caused by the
redistribution of noncovalent bonds. Differential scanning calorimeters measure this heat uptake.
Concerning NanoDSF, this technique records the intrinsic
fluorescence of Tryptophan (Trp) and Tyrosine (Tyr) residues,
which are very sensitive to changes in their local environment.
Thermal unfolding is measured by monitoring the intrinsic Trp
and Tyr fluorescence intensity, and the position of the emission
maximum as a function of temperature. The fluorescence intensity
ratio between 330 and 350 nm is defined as an empirical parameter
to monitor the evolution of the microenvironment of the aromatic
residues during protein denaturation throughout the temperature
increases. This ratio sharply increases/decreases during thermal
unfolding, allowing to determine a Tm value [35]. The applicability
of nanoDSF is highly dependent on the presence of Trp and Tyr in
the folded core of the PDZ that are exposed upon unfolding.
Moreover, it is necessary to exclude that the observed signal
changes are caused by aggregation, as this will also lead to variations
in the fluorophore environment. A back-scattering measurement
can also be performed to determine if aggregation occurs (before or
concomitantly with the denaturation). The characteristics of the
temperature gradient are essential as it is related to the activation
energy via the Arrhenius equation [6]. Typically, a heating rate of
1
C/min is applied. Overall, comparing the melting temperatures
(ΔT m ) in different buffer compositions allows researchers to define
the optimal buffer condition as an increase in Tm corresponds to a
better thermal stability and to a reduced conformational flexibility.
1.4.3 Nuclear Magnetic
Resonance (NMR)
NMR is based on the measure of the absorption of radiofrequency
(RF) radiation by an atomic nucleus located in a strong magnetic
field. The principle of NMR is that atomic nuclei, with an odd
number of protons (
1 H,
13
C,
15
N,
31
P, . . .), neutrons, or both,
have an intrinsic nuclear spin. When an atomic nucleus with a
nonzero spin is placed in a magnetic field, the nuclear spin aligned
in the same direction or in the opposite direction to the field.
Different energies characterize these two types of nuclear spin
alignment, and the application of a magnetic field facilitates the
degeneration of nuclear spins. An atomic nucleus whose spin is
aligned with the field will have less energy than when its spin is
aligned in the opposite direction of the field.
The energy of an NMR transition depends on the magnetic
field strength as well as on the proportionality factor applied to each
nucleus called the gyromagnetic ratio. The local environment
100
Ce ´ lia Caillet-Saguy et al.