Membrane-Embedded Channel of Bacteriophage Phi29 DNA-Packaging Motor for Translocation and Sensing of Double-Stranded DNA - Nanopores: Sensing and Fundamental Biological Interactions - page 98

Fig. 4.13 One way-traffic in DNA translocation through a connector channel reflected in the
change of DNA translocation frequency related to the orientation of connectors in bilayers with
multiple connector insertions. Various concentrations of DNA were premixed in both chambers
before applying the voltage. Insert: For the individual experiments (a-d), the DNA translocation
frequencies after the insertion of each connector channel in the BLM are shown.
Figures reproduced with permissions from: Ref. [ 44 ], # American Chemical Society

(Fig. 4.13a, c, d ). When one additional connector was inserted with an opposite
orientation that did not allow DNA to pass, the frequency of DNA trans-

location remained unchanged (Fig. 4.13a, b ).

4.6.4 The Orientation of the Connector Was Probed Using
Ni-NTA Nanogold Targeted at C-Terminal His-Tag

To elucidate the orientation of the connector in the BLM, reengineered connectors
containing a His-tag incorporated at the wider C-terminal end of each connector
subunit were used. The incorporation of the terminal His-tag did not affect
the folding or DNA packaging activity of the connector channels [ 42 ]. The conduc-
tion experiments were carried out under asymmetric ionic conditions (Fig 4.14 )[ 50 ].
A 1.8 nm Ni-NTA nanogold particle (150 pM) was used to bind to the His-tag at the
C-terminus. The experiment was designed such that the nanogold was premixed
with the buffer on one side of the chamber only. For instance, if the nanogold is
present in the trans- side under single or multiple connector channel insertion
conditions (Fig. 4.14 ), it will only bind to the connector with its His-tagged