Biomedical Engineering Reference
In-Depth Information
cond conductance states equals 78 % in the presence, and only 43
% in the absence, of NiCl
2
. Only 7 % of all events were found with
a conductance level above 600 pS. From these findings, it can be
concluded that the complexation of bipyridine by Ni
2+
indeed re-
sults in a considerable confinement of the observed multiple con-
ductance states. Assuming that the lowest conductance state is a
result of the formation of a six helix bundle, helix bundles with six
and seven helices are preferentially formed in the presence of Ni
2+
,
while higher-order aggregates become less likely.
2. Protein Channels
Relatively large transmembrane proteins such as Į-hemolysin
63
and the glutamate receptor
19
have been reconstituted into mem-
branes on orifices in either polycarbonates or silicon, with diame-
ters greater than 1 μm. We raised the question of how the pore size
of the porous material limits the incorporation and the functionali-
ty of complex integral membrane proteins in nano-BLMs. To ad-
dress this question, the functional insertion of the outer membrane
protein F (OmpF) from
E. coli
and of the eukaryotic protein con-
nexon 26 (Cx26) was investigated in nano-BLMs on porous alu-
mina with pore diameters of 60 nm by observing the ion channel
activity in voltage clamp experiments, as detailed below.
(
i
)
Outer Membrane Protein F
The porin OmpF is a well characterized protein in terms of
structure
64,65
and channel activity. It is composed of 16 antiparallel
aligned
E
-sheets (
E
-barrel), connected by amino acid sequences
building up a water-filled pore. Three of these monomeric units
with a molecular weight of 37.1 kDa and a length of 5 nm are ar-
ranged around a three-fold molecular axis. One protein covers an
area of roughly 80 nm
2
. In a nano-BLM, it is surrounded by just a
few thousand lipids. Ion selectivity and conductivity of the channel
is assumed to be a result of the constriction zone
66
that is formed
by loop 3, which folds into the barrel at approximately half the
height of the channel. By voltage clamp experiments, we were able
to prove that the protein is fully functional in nano-BLMs. The in-
sertion of the porin results in a three-step increase and decrease in
current due to the stepwise opening and closing of each subunit
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