Biomedical Engineering Reference
In-Depth Information
Fig. 4.14 Probing the orientation of the connector using Ni-NTA Nanogold targeted at C-terminal
His-tag. (a, c,ande) Illustration of strategy for orientation probing with equal concentration of
dsDNA added to both
cis
-and
trans
-chamber and same amount of Ni-NTA Nangold for each
experiment. (b) A 30-min current trace showing the translocation of dsDNA without blocking by
Ni-NTANanogold added to the
trans
-chamber; (d) A current trace showing three binding steps of Ni-
NTANangold to C-His connector after addition of Ni-NTA to the bottom
trans
-chamber. (f) A current
trace showing the translocation of dsDNA and blocking of DNA translocation by Ni-NTA Nanogold
added to the top
cis
-chamber. For all these experiments, a
trans
-membrane potential of
75 mV was
applied. Figures reproduced with permissions from: Ref. [
44
],
American Chemical Society
#
C-terminal end oriented towards the
trans
-chamber (Fig.
4.14c
), and will not bind to
connectors with the N-terminus facing the
trans-
side (Fig.
4.14a
). Discrete stepwise
blockage of the channel with a corresponding decrease in conduction was observed
when a single connector with appropriate orientation for nanogold binding was
present (Fig.
4.14d
). The binding of each nanogold resulted in uniform ~30%
blockage in current (Fig.
4.14d
). The same results were obtained in the case of
membranes with multiple channels (data not shown).
The same concentration of dsDNA was added in both sides of the chamber to
aid the appraisal of the DNA translocation direction, since dsDNA only migrates
from the negative towards the positive potential due to its negatively-charged
phosphate backbone. Based on the placement of the nanogold in either the
cis
-or
trans-
side of the chamber (experimental design shown in Fig.
4.14a, c, e
) under a
constant negative
trans
-membrane potential,
the orientation of the connector
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