Nanopore Force Spectroscopy: Insights from Molecular Dynamics Simulations - Nanopores: Sensing and Fundamental Biological Interactions - page 343

Fig. 14.2a . The tilt of the protein was accompanied by a bend in the DNA greater
than 60 ∘ , which rotated the portion of the molecule above the enzyme towards the
membrane. Such a sharp bend over just a few tens of basepairs would be difficult
with B-form dsDNA, which has a persistence length of about 50 nm; however, at
2.0 V, the DNA below the enzyme was stretched into an extended form with only
loose coupling between the strands. The simulations also showed that, during
rupture, one strand leaves the binding site first, followed shortly by the other.
Our definition of the threshold voltage as the voltage at which the mean time
until rupture is much greater than the timescale of the experiment make its estima-
tion difficult for MD because the timescale accessible to MD is much less than that
accessible to experiments. However, MD can give an upper bound for the threshold
voltage that, due to the sharpness of the dependence of the rupture rate on the
threshold voltage, comes quite close to the experimental value. Figure 14.2b shows
that the time until rupture rises sharply as the transmembrane voltage approaches
2.0V. At 4.0V, rupture occurs in only 3.4 1nswererequired

for rupture to occur. Decreasing the transmembrane voltage by just 0.5V (to 2.0V)
increased the rupture time to 75

0.1 ns. At 2.5V, 20

1 ns. These results bear some resemblance to
experiments, where threshold voltage for rupture of an Eco RI-DNA complex in
a pore of a similar geometry was shown to lie between 1.5 and 2.0V [ 22 ].
MD also permits qualitative comparison of the rupture kinetics between differ-
ent systems. For example, the rupture kinetics of different enzymes bound to DNA
or the same enzyme bound to different sequences could be compared. Note that
comparisons of the kinetics among enzymes bound to different DNA sequences
cannot be accomplished by simply mutating bases in the computational model.
Crystallography revealed that the conformation of Bam HI (an enzyme with an
active site very similar to that of Eco RI) is markedly different when it is specifically
bound to its cognate sequence than when it is nonspecifically bound to a sequence
that differed by a single basepair [ 43 ]. Hence, an experimentally derived structure
containing the DNA sequence of interest is required because the necessary changes
to the enzyme conformation are unlikely to occur on the timescale accessible to MD
simulations if the simulation is begun from a generic structure.
In NFS experiments one can measure the transmembrane voltage required for
rupture, but not the force on the complex at rupture. Thus, using MD, we embarked
on two approaches to determine the force on the complex during the experiments. In
the first approach, we extracted the force directly from simulations that mimicked
experimental conditions [ 22 ]. This approach was complicated by the presence of
solvent mediated forces, hydrodynamic effects, and artificial forces due to the
thermostat. However, rupture forces ranging from 1,600 to 2,800 pN were recorded
for simulations performed at 3.0 and 4.0 V.
In the second approach, we applied a methodology more akin to that used in
optical tweezers, although with a number of conveniences not available in experi-
ment. Here, a portion of the DNA was attached to one end of a virtual spring while
the other end was displaced at various rates. The restriction enzyme was also
restrained to its initial position by virtual springs; thus, the displacement of the
spring end eventually led to rupture of the complex. Protocols such as these are