Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 100

embedded in the membrane could be determined. It was found that dsDNA
translocated one way from the N-terminal narrow end toward the C-terminal wide
end. For example, when nanogold was placed in the bottom trans -chamber,
nanogold binding was not observed while dsDNA translocation occurred,
indicating that in this case, the C-terminus containing the His-tag was facing the
upper cis -chamber and the dsDNA translocated from the N-terminus at the bottom
trans -chamber that was negative (Fig. 4.14b ). Under the same conditions, in the
other case where the connector is oriented such that the C-terminus is facing the
trans -chamber, stepwise binding of nanogold was observed without any DNA
translocation (Fig. 4.14d ). In another scenario, when the nanogold was added to
the upper cis -chamber, dsDNA translocation occurred initially and was subse-
quently blocked, indicating that in this case the His-tagged C-terminus was facing
the upper cis -chamber, where nanogold binding occurs, and dsDNA translocated
from the N-terminus facing the bottom trans -chamber (Fig. 4.14f ).
4.7 Applications of Phi29 Motor Channel
and Future Perspectives
The ingenious, intricate and elegant design of the connector and its channel
motivated its application in nanotechnology. A major concern in using protein
nanopores relates to their soft nature, fluctuations in folding, dynamics in structure,
and the reproducibility of the signal generated. We demonstrated that the phi29
connector channels overcome these aforementioned limitations. The channels
are robust and stable under a wide range of experimental conditions. The conduc-
tance is uniform, demonstrating a perfect linear relationship with respect to
the applied voltages and does not display voltage gating properties under the
reported conditions. The dsDNA channel blockade events are nearly identical.
By virtue of its channel size and well-behaved properties, the phi29 connector is
ideally suited for reengineering a system that can operate outside its natural
environment and has tremendous potential to impact biology, engineering, medi-
cine, and other nanotechnological fields.
4.7.1 A Novel System for Studying the Mechanism
of Viral DNA Packaging
The work reported here represents a new system for understanding the mechanism
underlying DNA translocation through the motor channel in viral DNA packaging.
While the one-way traffic phenomenon in the lipid membrane embedded
system is a passive transport process mediated by electric potential, it would be
worthwhile exploring whether the active motor also exercises a one-way traffic
mechanism [ 44 ].