Biomedical Engineering Reference
In-Depth Information
mdm2-mediated inactivation. The DFS investigation of the interaction of p53 with
mdm2 was done by using, for the first time, full-length proteins for both p53 and
mdm2 (Funari et al., 2010); previous studies by bulk techniques having been car-
ried out using only partial domains of both proteins (Sch on et al., 2002). Funari
et al. (2010) found a single linear trend in the unbinding force versus the logarithm
of the loading rate; the corresponding dissociation rate
k
off
of 1.5 s
−
1
is indicative
of a transient character. Interestingly, this result, obtained at single-molecule level
and using full-length proteins, is in a good agreement with that obtained in bulk by
partial chains of both the proteins (1
1
;Schon et al., 2002; Domenici et al.,
2011). Notably, a comparison of measurements by DFS using full length or por-
tions of biomolecules makes it possible to obtain information on which regions are
involved in the interaction and even on the interplay among the different regions on
the kinetic properties. Bizzarri and Cannistraro have analyzed the DFS data of the
mdm2-p53 complex in the framework of the Jarzinski model by separately evaluating
the contribution to the total binding free energy arising from both the stretching of
the linker used in the setup and the complex unbinding process (Bizzarri and Cannis-
traro, 2011). The extracted unbinding free energy of
2s
−
−
8.4 kcal/mol has been found to
be in a good agreement with that measured in bulk by isothermal titration calorime-
try again using partial domains of both the proteins (from
−
6.6 kcal/mol;
Sch on et al., 2002). Starting from the evidence that azurin was able to promote an
anticancer activity through its binding to p53
in vitro
and
in vivo
, the interaction
between azurin and p53 was studied by DFS (Taranta et al., 2008). From the analysis
of the unbinding force as a function of the logarithm of the loading rate, Taranta et al.
found a single barrier in the energy landscape with a dissociation rate of 0.09 s
−
1
(see Table 6.3). This result, indicating a lifetime of about 10 s, is consistent with the
formation of a relatively stable complex. With the aim to extract some information
on the interaction sites between azurin and p53, the DFS study has been comple-
mented by computational docking. The possible binding regions between azurin and
two different partial domains of p53 (the DNA binding domain and the N-terminal
domain) have been proposed and refined in connection with the available mutagene-
sis data (De Grandis et al., 2007; Taranta et al., 2009). These structural informations
were found to be extremely insightful to design azurin-derived peptides retaining the
same ability of azurin to penetrate cancerous cells and to extert a strong anticancer
activity with minimal side effects (Yamada et al., 2009).
Although DFS has been mainly applied to investigate bimolecular complexes, the
setup can be easily adapted to study ternary complexes or even to perform compet-
itive binding experiments. Recently, a possible competition of azurin with mdm2
for the same binding site on p53 has been carried out by conceiving a competitive
blocking experiments, according to the strategy sketched in Figure 6.8. First, the fre-
quency of the unbinding events between p53 immobilized on the substrate and azurin
anchored to the tip has been estimated before and after blocking the substrate with
a solution of free azurin; a significant decrease in the unbinding frequency being
observed upon azurin addition. The experiment has been then repeated by blocking
the substrate with a solution of free mdm2. Successively, the experiment has been
−
8.8 to
−
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