denaturation. This approach, developed by Pantoliano et al. in
2001 [30], has been originally registered under the trademark
“Thermofluor,” a name now used as a synonym for all such extrinsic
dye-based experiments. Ideally, the rapid increase in the proportion
of unfolded protein will lead to a sharp increase of the fluorescent
signal over a short temperature range, generating a sharp sigmoidal
curve of fluorescence and again allowing the calculation of T m from
the maximum of the first derivative. These measurements can be
carried out in multiwell plates in a quantitative polymerase chain
reaction (qPCR) machine. This approach is very popular and accessible because of the wide availability and relatively low cost of such
instruments. However, there are some drawbacks vs. the intrinsic
approach: (1) the addition of the extrinsic dye modifies the buffer
composition, (2) some dyes may be incompatible with some buffers, and (3) binding of the dye may itself destabilize the protein.
An exhaustive review on Thermofluor has been written by Boivin
and coworkers [31].
An alternative is DSC, the only direct technique to study the
thermodynamics of protein thermal stability. Its advantages are that
no modification of the protein or any additional component is
required. On the negative side, DSC is typically both more timeand sample-consuming than DSF of intrinsic fluorophores or Thermofluor [32]. However, automated DSC machines are becoming
more widely available, reducing the time required for the
experiments.
DSC measures the molar heat capacity C p of the sample as a
function of temperature T and allows the determination of the
change in enthalpy and entropy upon denaturation. Folded and
unfolded states have different heat capacities (largely due to their
different interactions with water molecules), and changes in populations of the two states as the temperature is increased mean that a
peak appears in the C p vs. T plot with its maximum at T m and its
integral corresponding to the change in enthalpy upon denaturation. As DSC directly measures the heat capacity, it is very well
suited to measure the protein activation energy that is directly
correlated to thermal flexibility [33]. The protein activation energy
corresponds to the energy barrier that needs to be passed to allow
unfolding.
1.4.2 Assessing Colloidal
Stability/Aggregation
DLS is the method of choice to detect the formation of protein aggregates and to follow the effects of changes in the experimental protocols to improve solubility. A variety of changes can be
made either upstream to/during the production/purification process (for details see Refs. 26 and 34 and Note 4), or to buffer
conditions. Many groups use DLS as a technique to improve the
solution conditions of their proteins, in particular before crystallization studies [35, 36]. Recent DLS instrumental developments
18
Bertrand Raynal et al.
2001 [30], has been originally registered under the trademark
“Thermofluor,” a name now used as a synonym for all such extrinsic
dye-based experiments. Ideally, the rapid increase in the proportion
of unfolded protein will lead to a sharp increase of the fluorescent
signal over a short temperature range, generating a sharp sigmoidal
curve of fluorescence and again allowing the calculation of T m from
the maximum of the first derivative. These measurements can be
carried out in multiwell plates in a quantitative polymerase chain
reaction (qPCR) machine. This approach is very popular and accessible because of the wide availability and relatively low cost of such
instruments. However, there are some drawbacks vs. the intrinsic
approach: (1) the addition of the extrinsic dye modifies the buffer
composition, (2) some dyes may be incompatible with some buffers, and (3) binding of the dye may itself destabilize the protein.
An exhaustive review on Thermofluor has been written by Boivin
and coworkers [31].
An alternative is DSC, the only direct technique to study the
thermodynamics of protein thermal stability. Its advantages are that
no modification of the protein or any additional component is
required. On the negative side, DSC is typically both more timeand sample-consuming than DSF of intrinsic fluorophores or Thermofluor [32]. However, automated DSC machines are becoming
more widely available, reducing the time required for the
experiments.
DSC measures the molar heat capacity C p of the sample as a
function of temperature T and allows the determination of the
change in enthalpy and entropy upon denaturation. Folded and
unfolded states have different heat capacities (largely due to their
different interactions with water molecules), and changes in populations of the two states as the temperature is increased mean that a
peak appears in the C p vs. T plot with its maximum at T m and its
integral corresponding to the change in enthalpy upon denaturation. As DSC directly measures the heat capacity, it is very well
suited to measure the protein activation energy that is directly
correlated to thermal flexibility [33]. The protein activation energy
corresponds to the energy barrier that needs to be passed to allow
unfolding.
1.4.2 Assessing Colloidal
Stability/Aggregation
DLS is the method of choice to detect the formation of protein aggregates and to follow the effects of changes in the experimental protocols to improve solubility. A variety of changes can be
made either upstream to/during the production/purification process (for details see Refs. 26 and 34 and Note 4), or to buffer
conditions. Many groups use DLS as a technique to improve the
solution conditions of their proteins, in particular before crystallization studies [35, 36]. Recent DLS instrumental developments
18
Bertrand Raynal et al.