Biomedical Engineering Reference
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holds, which reproduces the photocurrent quite well. Our results
resemble those found by Bamberg et al.,
91
who added bR-doped
liposomes to a freestanding lipid bilayer. They also allocated a
non-zero stationary photocurrent in the absence of a proton decou-
pler to the activity of integrated bR.
Independent of the scenario found on the porous surface, the
proton pump bR is actively reconstituted. As the proton transport
of bR is unidirectional from the C- to the N-terminus, only the net
current is monitored as a result of an excess orientation of the pro-
teins in one direction. Thus, we can state that more than 50 % are
directed with the C-terminus towards the
cis
side of the membrane.
It remains, however, unclear how many proteins have been recon-
stituted. It is known that, by using the reversed phase method,
103,105
bR is predominantly reconstituted in liposomes with the
C-terminus facing the outer membrane leaflet. If one assumes that
the direction of integration is preserved, when bR is transferred
from the proteoliposomes to the pore-spanning bilayer, a mecha-
nism for liposome spreading can be suggested. Whereas on hydro-
philic supports liposomes unroll in a fashion that renders the inner
leaflet of the membrane facing upwards, on hydrophobic materials
the inner membrane leaflet of liposomes is directed downwards.
39
From the photocurrent results we conclude that liposomes spread-
ing on CPEO3-functionalized porous alumina spread in a way that
is described for vesicles on hydrophobic surfaces.
To investigate the net orientation, one could make use of lan-
thanium cations, which interact with bR at its C-terminus and
block the proton pump activity.
106
From the remaining signal, the
orientation of the molecules could be estimated. Considering a
turnover rate of 100 protons s
-1
, approximately 6·10
7
molecules
contribute to the signal displayed in
Fig. 21 A
. With a protein area
of 11.5 nm
2
, 0.06 % of the electrically active area would be cov-
ered by protein. This area fraction is significantly smaller than
what is expected if all proteins are transferred from the liposomes
containing 1 mol% bR to the pore-suspending bilayer.
Another interesting aspect is the susceptibility of pore-
spanning membranes to the protonophor CCCP. While, in case of
nano-BLMs, up to 40 μM CCCP was added to obtain a significant
increase in stationary current, only 2 μM is required to significant-
ly increase the stationary current during the illumination process.
This result is in line with the notion that pore-spanning membranes
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