Biomedical Engineering Reference
In-Depth Information
that the unbinding force as a function of the logarithm of the loading rate is char-
acterized by a linear trend but with different slope and intercept parameters for the
two systems (see Table 6.3). A dissociation rate of 6.3 s
−
1
observed for the binary
complex is an order of magnitude lower than that of the ternary complex (0.5 s
−
1
).
The higher stability shown by the ternary complex supports the important role of the
synaptosome protein to allow positioning of vesicles at a maximal distance (about
12 nm) from the plasma membrane for a longer time than that corresponding to the
binary complex. These findings concord with other results from other techniques and
confirm a previous so-called zippering model taking into account the formation of
coiled coils in the binary complex.
Successively, the analysis of the DFS data on the syntaxin-synaptobrevin binary
complex has been extended within the framework of the very interesting Jarzyniski
theoretical model (Liu et al., 2008). It allows to determine the equilibrium binding
free energy of the complex from the work done by the applied force along several
nonequilibrium paths connecting the initial and the final states of a reaction (see
Chapter 3; Hummer and Szabo, 2005; Jarzynski, 1997). A simplified expression has
been worked out for the binding free energy Δ
G
:
N
i
=
1
1
N
e
−
W
i
/
k
B
T
e
−
Δ
G
/
k
B
T
=
(6.4)
where
N
is the number of independent iterations of the unbinding process, and
W
i
is the work along the
i
th unbinding path done under the application of the exter-
nal force. For a given force curve, the work done by the applied force during the
unbinding process can be determined by calculating the integral from the beginning
of the nonlinear course in the retraction curve up to the end of the jump-off event (see
the inset of Figure 6.2). Accordingly, they have estimated a binding free energy for
the syntaxin-synaptobrevin complex of about (49
k
B
T
. Such a value has been
compared with that evaluated from the Arrhenius relationship (
k
off
∝ e
−
(
−
±
5
)
Δ
G
∗
/
k
B
T
)
)
at three different temperatures (
T
= 277, 287, 297 K) obtaining Δ
G
k
B
T
.
The discrepancy between the two Δ
G
values has been traced back to the fact that the
applied force does not act along the bond axis, making in this case the application
of the Bell-Evans model not completely appropriate. This approach, remarkably,
endows DFS experiments with the capability to measure the binding free energy,
besides determining
k
off
and
x
β
.
Recently, DFS has been applied to investigate complexes involving the human
tumour suppressor p53, which is a protein known to play a crucial role in trigger-
ing cancer defense mechanisms. In the presence of different stress signals, p53 is
stabilized through posttranslational modifications, its cellular levels increase, and it
can induce the expression of target genes that, in turn, control the process of DNA-
repair, the cell-cycle arrest, and the apoptotic cascade (Vogelstein et al., 2000). The
activity of p53 is downregulated by the mdm2 oncogene that promotes its ubiquitin-
dependent degradation through the formation of a complex with it (Chene, 2004).
On such a basis, the mdm2-p53 complex is a preferential target for anticancer drug
design devoted to restore normal p53 function in tumour cells by preventing its
=(
33
±
6
)
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