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& Dasgupta, 2008; Domadia, Swarup, Bhunia, Sivaraman, &Dasgupta, 2007
). It has
also been used to characterize the binding of modulators of microtubule assembly on
tubulin (
Banerjee et al., 2005; Das et al., 2009; Gupta et al., 2003; Menendez,
Laynez, Medrano, & Andreu, 1989; Rappl et al., 2006; Tsvetkov et al., 2011
), to
study stathmin-tubulin binding (
Honnappa et al., 2003
) and more recently tau-
tubulin binding (
Tsvetkov et al., 2012
).
18.3.1
Stathmin-tubulin interaction
In 2003, Steinmetz and coauthors from Paul Scherrer Institute published an extensive
characterization of the thermodynamics of the stathmin-tubulin interaction
(
Honnappa et al., 2003
). They determined the stoichiometry, binding constant, var-
iation of enthalpy and of entropy under different conditions of pH, temperature, and
nucleotide presence (GTP/GDP). Under all investigated conditions, they obtained
simple sigmoid binding isotherms, which can be well fitted with a simple one-set-
of-sites binding model, described by following equations:
þ
TS
K
0
; D
ð
H
0
Þ
T
S
>
(18.1)
þ
T2S
K
0
; D
ð
H
0
Þ
T
TS
>
They reported two binding sites of equal affinity with an equilibrium binding con-
stant of
K
0
¼
10
6
M
1
6.0
and large negative molar heat capacity change
860 cal mol
1
K
1
), which suggest that the major driving force of the
binding reaction was hydrophobic interactions (
Fig. 18.4
). Nevertheless, earlier stud-
ies using several techniques, including pull-down assays (
Holmfeldt et al., 2001;
Larsson et al., 1999
) and analytical ultracentrifugation (
Amayed, Carlier, &
Pantaloni, 2000; Jourdain, Curmi, Sobel, Pantaloni, & Carlier, 1997
), suggested
the existence of two highly cooperative binding sites. These findings led Honnappa
and coauthors to conclude that ITC data contrasted with earlier studies proposing that
the second tubulin subunit is bound distinctly tighter than the first one. Nevertheless,
several models can fit the same curve. Indeed, the fact that ITC titration results in
a simple thermogram does not guarantee that the simplest model is the real one.
In other words, in this case, the principle of Occam's razor could be summarized
as “other things being equal, a simpler explanation is better than a more complex
one.” As mentioned above, the sigmoid form of binding isotherm could also be
observed for more complex models in the case of degenerate parameters. For exam-
ple, for a model of nonequal interacting sites (
Fig. 18.6
) described by the following
equations
(
D
C
p
¼
þ
TS
K
A
1
; D
ð
H
A
1
Þ
T
S
>
þ
T2S
K
A
2
; D
ð
H
A
2
Þ
T
TS
>
(18.2)
þ
ST
K
B
1
; D
ð
H
B
1
Þ
S
T
>
ST
þ
T
T2S
K
B
2
; D
ð
H
B
2
Þ
>
if there is strong cooperativity (
K
A
2
K
A
1
and
K
B
2
K
B
1
) between two equal sites
(
D
H
A
1
¼D
H
B
1
,
K
A
1
¼
K
B
1
) and if binding of first ligand molecule does not change the
