Nanopore Force Spectroscopy: Insights from Molecular Dynamics Simulations - Nanopores: Sensing and Fundamental Biological Interactions - page 342

Fig. 14.2 MD simulations of a restriction enzyme-DNA complex interacting with a nanopore.
(a) Snapshots from an MD simulation in which an external voltage of 4.0 V is applied across the
membrane. The membrane has a thickness of 10 nm and the minimum diameter of the pore is
2.9 nm. The elapsed time since the application of the voltage is shown above each snapshot.
(b) Rupture of the Eco RI-DNA molecular bond at various applied voltages. To characterize the
separation between the enzyme and DNA, we plot the distance between the phosphate of the DNA
adjacent to the bond cleaved by Eco RI (when Mg 2+ is present) and the glutamic acid in the active
site of Eco RI [ 37 ]. Due to the symmetry of the Eco RI dimer, there are two active sites; the data
plotted here are for the site nearest the pore. Abrupt increases in this distance are observed when
the enzyme and DNA dissociate. In all cases, a force was applied to reduce the affinity of the DNA
for the pore walls [ 20 ]; however, for the 2 V simulation this force was not applied until t ¼ 56 nm.

penetrate into the pore and how was it positioned in the pore when force was
applied? How did the conformation of the enzyme and DNA change under force?
How did rupture occur? Did the DNA simply slip through the binding site? Or did the
conformation of the protein change considerably? What was the force at rupture?
Which parts of the DNA decoupled from the protein first? MD simulations, one of
which is illustrated in Fig. 14.2a , provided answers to all these questions.
The simulations immediately yielded qualitative insights. As can be seen from
Fig. 14.2a , the enzyme did not completely enter the pores, even under a high force.
Under load, however, the enzyme showed a tendency to tilt, allowing one lobe to
penetrate the pore opening, as shown clearly in the 3 and 6 ns snapshots shown in