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between
-tubulin and the N-terminal cap of stathmin. In this context, whichever model
is hypothesized, only the overall constant of formation of T2S complex (
K
T2S
)canbe
determined via
K
0
,as
K
A
1
,
K
A
2
,
K
B
1
,or
K
B
2
cannot be determined from the model
of nonequal interacting sites if the binding isotherm is a degenerate sigmoid curve.
Measurement of
K
0
or further extrapolation to
K
T2S
enables only the characterization
of T2S complex formation (described by Eq.
18.3
) with values of stoichiometry, en-
tropy, enthalpy, and free energy. The knowledge of these thermodynamic parameters
allows one to characterize the nature of the forces involved in the interaction. In the
example presented in
Fig. 18.4
, below 28
C, since
a
0(
Fig. 18.4
), the only driving
force of T2S complex formation is hydrophobic interactions (
D
H
>
D
S
>
0), whereas above
28
C, the reaction is enthalpy (
0). Rather than the
intrinsic values of these parameters, which can vary greatly depending on the buffer
conditions and temperature, it is the comparison of the parameters obtained in different
conditions that will bring new information about the interaction. And despite the open
question about the true nature (cooperative vs. noncooperative) of stathmin-tubulin
binding, ITC enabled investigators to quantify the impact of each one of the four stath-
min phosphorylations and different combinations of them, on its affinity for tubulin
(
Honnappa, Jahnke, Seelig, & Steinmetz, 2006
). It enabled the authors to provide
in
vitro
the biophysical basis for understanding the mechanismbywhich stathmin activity
gradients will regulate local microtubule growth. This approach has also been used to
determine the consequences of the presence of anticancer agents, such as vinblastine, on
the activity of stathmin (
Devred et al., 2008
). Comparison of stathmin-tubulin binding
in the presence or absence of vinblastine revealed an increase in the stathmin affinity
for tubulin in the presence of vinblastine, setting themolecular basis of a new or revised
mechanism of action of this MTA.
D
H
<
0) and entropy driven (
D
S
>
18.3.2
Tau-tubulin
ITC has also been used to study the interaction of tau with tubulin. We can expect
that the binding of a stabilizing MAP such as tau, whose individual repeat domains
can bind and stabilize microtubules (
Aizawa et al., 1989; Butner & Kirschner,
1991; Devred, Douillard, Briand, & Peyrot, 2002; Ennulat, Liem, Hashim, &
Shelanski, 1989; Goedert, Wischik, Crowther, Walker, & Klug, 1988; Gustke,
Trinczek, Biernat, Mandelkow, & Mandelkow, 1994
), is more complex to study
than the binding of a destablizer, such as stathmin. The presence of any factor that
favors or inhibits tubulin polymerization may have an impact on the extent of the
tau-induced self-assembly and potentially on the thermodynamic parameters deter-
mined. This is why the recent ITC study of tau-tubulin interaction was conducted
in a minimum phosphate-GTP buffer in the absence of Mg
2
รพ
(
Tsvetkov et al.,
2012
). Even though tau has been studied for more than 40 years, very little is
known about its structure (
Harbison, Bhattacharya, & Eliezer, 2012
). In addition,
there are several discrepancies regarding its mode and parameters of binding to
tubulin, probably in part due to the fact that microtubules can induce the formation
of tau filaments (
Duan & Goodson, 2012
). Nevertheless, several studies have
