report the analyses by MST of the same interaction using DNA
labeled with a fluorescent probe. We previously used ITC to characterize protein–protein and protein–DNA interactions involved in
the NHEJ pathway [6, 11, 12] and in other DNA metabolism
pathways [13–15].
Isothermal titration calorimetry measures the heat generated or
consumed upon the interaction of two molecules, called hereafter
the receptor and the ligand [16, 17]. The molecular interaction
between the receptor and the ligand can be defined by the following equation: ΔG
¼ ÀRT lnK a ¼ RT lnK d ¼ ΔH – TΔS, where ΔG
(kcal/M) is the standard Gibbs free energy, R (kcal/M) the gas
constant, T (K) the temperature, K a (M
À1 ) the equilibrium association constant, and K d (M) the equilibrium dissociation constant.
The second part indicates that the free energy is the sum of an
enthalpy term ΔH (kcal/M) and an entropic term ÀTΔS (kcal/M).
Figure 1 represents a schema of the calorimeter. A constant power is
applied to the reference cell. A very small temperature difference is
maintained between the sample and reference cells thanks to a
feedback circuit that applies a variable power to the sample cell.
The injection of a small volume of ligand, loaded in the syringe,
leads to a heat exchange when the ligand interacts with the receptor. Exothermic reactions generate heat and induce a decrease in
the feedback power. Endothermic reactions absorb heat and lead to
an increase of the feedback power. The instrument modifies the
power applied to the measurement cell according to the nature of
the interaction. If the reaction is exothermic, it reduces the power,
and we observe a negative peak in the thermogram (Fig. 1b, top).
On the contrary, if the reaction is endothermic, it increases the
power, and we observe a positive peak in the thermogram (as in
the Ku–DNA example presented here, Fig. 3b). The isotherm of
titration (Fig. 1b, bottom) is determined by integrating each peak
of the thermogram and by reporting the calculated values that
correspond to the enthalpy of the reaction. The x axis of the
isotherm is defined as the ratio of ligand/receptor molecules at
each step of the titration. The curve that best fits these points
gives, in the case of a single binding site, the ΔH, the K d , and the
stoichiometry of the interaction (Fig. 1b, bottom). ITC has several
advantages: (1) it is widely used since limited optimization is
needed; (2) the interaction is performed without labeling the partners; (3) both partners are in solution at opposite to methods where
one partner is linked to a surface (like SPR, BLI, switchSENSE);
(4) it is nondestructive, the complex receptor–ligand can be used
for other application (cristallisation for example) or re-purified on a
size exclusion chromatography or anion/cation exchange chromatography after measurement to separate the receptor and the
ligand; (5) it has no molecular weight limitation. This method
also has several limitations: (1) it consumes important quantities
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
127
labeled with a fluorescent probe. We previously used ITC to characterize protein–protein and protein–DNA interactions involved in
the NHEJ pathway [6, 11, 12] and in other DNA metabolism
pathways [13–15].
Isothermal titration calorimetry measures the heat generated or
consumed upon the interaction of two molecules, called hereafter
the receptor and the ligand [16, 17]. The molecular interaction
between the receptor and the ligand can be defined by the following equation: ΔG
¼ ÀRT lnK a ¼ RT lnK d ¼ ΔH – TΔS, where ΔG
(kcal/M) is the standard Gibbs free energy, R (kcal/M) the gas
constant, T (K) the temperature, K a (M
À1 ) the equilibrium association constant, and K d (M) the equilibrium dissociation constant.
The second part indicates that the free energy is the sum of an
enthalpy term ΔH (kcal/M) and an entropic term ÀTΔS (kcal/M).
Figure 1 represents a schema of the calorimeter. A constant power is
applied to the reference cell. A very small temperature difference is
maintained between the sample and reference cells thanks to a
feedback circuit that applies a variable power to the sample cell.
The injection of a small volume of ligand, loaded in the syringe,
leads to a heat exchange when the ligand interacts with the receptor. Exothermic reactions generate heat and induce a decrease in
the feedback power. Endothermic reactions absorb heat and lead to
an increase of the feedback power. The instrument modifies the
power applied to the measurement cell according to the nature of
the interaction. If the reaction is exothermic, it reduces the power,
and we observe a negative peak in the thermogram (Fig. 1b, top).
On the contrary, if the reaction is endothermic, it increases the
power, and we observe a positive peak in the thermogram (as in
the Ku–DNA example presented here, Fig. 3b). The isotherm of
titration (Fig. 1b, bottom) is determined by integrating each peak
of the thermogram and by reporting the calculated values that
correspond to the enthalpy of the reaction. The x axis of the
isotherm is defined as the ratio of ligand/receptor molecules at
each step of the titration. The curve that best fits these points
gives, in the case of a single binding site, the ΔH, the K d , and the
stoichiometry of the interaction (Fig. 1b, bottom). ITC has several
advantages: (1) it is widely used since limited optimization is
needed; (2) the interaction is performed without labeling the partners; (3) both partners are in solution at opposite to methods where
one partner is linked to a surface (like SPR, BLI, switchSENSE);
(4) it is nondestructive, the complex receptor–ligand can be used
for other application (cristallisation for example) or re-purified on a
size exclusion chromatography or anion/cation exchange chromatography after measurement to separate the receptor and the
ligand; (5) it has no molecular weight limitation. This method
also has several limitations: (1) it consumes important quantities
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
127