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suggested the existence of two binding sites, one that may overlap the paclitaxel
binding site and that would be located in the lumen, and another one on the outside
wall of MT (
Ackmann, Wiech, & Mandelkow, 2000; Kar, Fan, Smith, Goedert, &
Amos, 2003; Makrides, Massie, Feinstein, & Lew, 2004
). These two sites would not
be equally accessible depending on the nature of experimental study, such as tau-
induced MT self-assembly versus tau binding to stabilized MT. ITC titration of tu-
bulin by tau results in a complex two-phase binding isotherm that could be well
fitted using two-sets-of-sites model, compatible with the two types of tau-tubulin
binding modes described in the literature: one corresponding to a high affinity bind-
ing site with a tau:tubulin stoichiometry of 0.2 and the other one to a low affinity
binding site with a stoichiometry of 0.8. Nevertheless, it cannot be excluded that
tau-tubulin binding follows a more complex model. To assign the real model, many
complementary experiments will need to be performed. Like in the case of stathmin-
tubulin interaction, even the simplest bindingmodel which resulted in the determination
of only apparent thermodynamic parameters helped us to gain new insights into the
mechanism of tau binding to tubulin. Indeed, even though tau induces the formation
of curved tubulin protofilament at 10
C, and the formation of microtubules at 37
C
(
Devred et al., 2004
;
Fig. 18.1
), tau-tubulin binding isotherm obtained at 10 and
37
Cwere both biphasicwith amaximumat a tau:tubulinmolar ratio of one, indicating
a similar bindingmodel (
Fig. 18.5
). The fact that tauwould bind similarly on anMT and
on a circular protofilament indicates that onMT the interaction is longitudinal (along the
same protofilament) and not transversal (bridging several parallel protofilaments). This
allowed us to rule out the models which hypothesized that tau stabilizes MT by binding
across several protofilaments on the MT lattice.
CONCLUSION
ITC is one of the latest and most powerful techniques to be used in characterizing the
binding affinity of ligands for proteins or proteins for proteins. But like most tech-
niques, it would be useless without other methods. ITC measures the heat exchange
and thus often relies on complementary studies to hint at or confirm what reaction is
really happening in the calorimetric cell. For example, analytical ultracentrifugation
is a technique of choice to determine stoichiometry, changes in conformation or
assembly state of the molecules studied (
Correia & Stafford, 2009; Demeler,
Brookes, & Nagel-Steger, 2009; Lebowitz, Lewis, & Schuck, 2002; Schuck,
2003
). Through the examples detailed in this chapter, we have shown that if a certain
number of precautions, due mostly to the nature of tubulin, are taken, ITC can be
used to thermodynamically characterize molecular interactions between tubulin
and MAPs. If stathmin binding to tubulin is now well characterized by ITC, there
is still a lot to understand about tau binding to tubulin. In summary, even though
the tubulin cytoskeleton is a challenging system to work on, ITC is a powerful tech-
nique able to provide significant advances in our understanding of tubulin interaction
with its partners.
