We analyzed the interactions of Ku with a 18 bp and a 42 bp
DNA duplex (Fig. 3b, c) and observed two endothermic reactions
(positive peaks) in both the cases. The interaction between Ku and
the 18 bp DNA shows a stoichiometry of 0.97 Æ 0.1 molecules of
18 bp DNA bound to one Ku, a K d of 3.5 Æ 0.8 nM, and an
μcal/sec
0.02
0.01
0.00
6.00
4.00
2.00
0.00
kcal.mol -1
of inj
-6 12 30 48 66 84
DNA
N
K D
(nM)
ΔH°
(kcal.mol -1 )
-TΔS°
(kcal.mol -1 )
ΔG°
(kcal.mol -1 )
18bp
0.97 ± 0.1 3.5 ± 0.8
5.1 ± 0.1
-16.6 ± 0.2
-11.5 ± 0.2
42bp
0.47 ± 0.1 2.4 ± 0.5
22.9 ± 0.3
-34.7 ± 0.5
-11.8 ± 0.1
B
18bp
C
Time (min)
Time (min)
Molar Ratio
0.0 0.5 1.0 1.5 2.0 2.5 3.0
D
+
+
18bp
42bp
A
66 84
C
n)
Time (min)
+
+
18bp
42bp
42bp
-6 12 30 48 66 84
kcal.mol -1
of inj
0.06
0.04
0.02
0.00
-0.02
μcal/sec
22.0
16.0
8.0
0.0
0.0 0.5 1.0 1.5 2.0
Molar Ratio
Fig. 3 ITC measurements between Ku and DNA. (a) Crystal structure of Ku-DNA complex with a 14 bp DNA. Ku
has a ring shape structure. The DNA present in the crystal structure has an additional hairpin part that has
been removed for clarity (pdb code 1JEY). A schema is presented of the two interactions analyzed with Ku and
either a 18 bp DNA or 42 bp DNA. (b) ITC measurement between Ku and a 18 bp DNA. The interaction is
endothermic with positive peaks on the thermogram. The heat exchanges observed in this interaction are very
weak (<0.02 μcal/s) and underline the high sensitivity of the VP-ITC. The isotherm of titration enables to
define ΔH, K d , and N of the reaction, reported in Table (d). One Ku molecule binds to an 18 bp DNA. (c) ITC
measurement between Ku and a 42 bp DNA hairpin. ΔH, K d , and N of the reaction are reported in Table (d).
Two Ku molecules bind on the 42 bp DNA leading to a stoichiometry N close to 0.5
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
135
DNA duplex (Fig. 3b, c) and observed two endothermic reactions
(positive peaks) in both the cases. The interaction between Ku and
the 18 bp DNA shows a stoichiometry of 0.97 Æ 0.1 molecules of
18 bp DNA bound to one Ku, a K d of 3.5 Æ 0.8 nM, and an
μcal/sec
0.02
0.01
0.00
6.00
4.00
2.00
0.00
kcal.mol -1
of inj
-6 12 30 48 66 84
DNA
N
K D
(nM)
ΔH°
(kcal.mol -1 )
-TΔS°
(kcal.mol -1 )
ΔG°
(kcal.mol -1 )
18bp
0.97 ± 0.1 3.5 ± 0.8
5.1 ± 0.1
-16.6 ± 0.2
-11.5 ± 0.2
42bp
0.47 ± 0.1 2.4 ± 0.5
22.9 ± 0.3
-34.7 ± 0.5
-11.8 ± 0.1
B
18bp
C
Time (min)
Time (min)
Molar Ratio
0.0 0.5 1.0 1.5 2.0 2.5 3.0
D
+
+
18bp
42bp
A
66 84
C
n)
Time (min)
+
+
18bp
42bp
42bp
-6 12 30 48 66 84
kcal.mol -1
of inj
0.06
0.04
0.02
0.00
-0.02
μcal/sec
22.0
16.0
8.0
0.0
0.0 0.5 1.0 1.5 2.0
Molar Ratio
Fig. 3 ITC measurements between Ku and DNA. (a) Crystal structure of Ku-DNA complex with a 14 bp DNA. Ku
has a ring shape structure. The DNA present in the crystal structure has an additional hairpin part that has
been removed for clarity (pdb code 1JEY). A schema is presented of the two interactions analyzed with Ku and
either a 18 bp DNA or 42 bp DNA. (b) ITC measurement between Ku and a 18 bp DNA. The interaction is
endothermic with positive peaks on the thermogram. The heat exchanges observed in this interaction are very
weak (<0.02 μcal/s) and underline the high sensitivity of the VP-ITC. The isotherm of titration enables to
define ΔH, K d , and N of the reaction, reported in Table (d). One Ku molecule binds to an 18 bp DNA. (c) ITC
measurement between Ku and a 42 bp DNA hairpin. ΔH, K d , and N of the reaction are reported in Table (d).
Two Ku molecules bind on the 42 bp DNA leading to a stoichiometry N close to 0.5
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
135