Biomedical Engineering Reference
In-Depth Information
layer, without the need for a spacious ionic reservoir beneath a
well-behaved lipid bilayer. In fact, their activation would cause an
increase in the proton concentration on top of the thiolipid mono-
layer (in the case of F
0
F
1
ATPase activated by ATP) or its decrease
(in the case of COX activated by ferrocytocrome
c
). In view of the
relative permeability of the leaky thiolipopeptide monolayers to
protons, this would determine an increase or a decrease in the pro-
ton electroreduction current on gold, as actually observed.
A tBLM consisting of a gold-supported thiolipopeptide, with a
soybean-PC monolayer on top, was used to incorporate the odorant
receptor OR5 from
Rattus norvegicus
during its
in vitro
synthe-
sis.
176
The vectorial insertion of the protein into the tBLM in a
functional and oriented form was verified. The incorporation and
orientation of the protein were shown by immunolabeling in com-
bination with surface plasmon enhanced fluorescence spectroscopy
(SPFS). Reversible ligand binding was demonstrated by surface-
enhanced infrared reflection absorption spectroscopy (SEIRAS).
Total internal fluorescence (TIRF) imaging, confocal fluorescence
correlation spectroscopy (FCS) and FRAP allowed the incorpora-
tion density and the translational mobility of OR5 to be quanti-
fied.
177
A gold-supported thiolipopeptide-based tBLM was also em-
ployed to incorporate the acetylcholine receptor (AChR), a ligand-
gated channel protein present in the postsynaptic membrane of the
muscle cell. The binding of acetylcholine to the receptor protein
causes a conformational change in the protein that allows the in-
flux of Na
+
ions into the muscle cell. The venoms of certain snakes
contain peptide neurotoxins (e.g., D-bungarotoxin) that are com-
petitive inhibitors of the AChR. The thickness of the lipid bilayer
of the tBLM, as estimated by SPR, increases from about 4.5 to
about 6.5 nm upon AchR incorporation.
178
This suggests a relative-
ly high packing density of the AchR molecules arranged perpen-
dicular to the bilayer. Addition of D-bungarotoxin causes a slow
but appreciable increase in thickness, thus denoting the binding of
this toxin to the binding sites of AChR, possibly accompanied by
major conformational changes. This test confirms that at least a
portion of the AchR protein is oriented with the extracellular do-
main pointing outside of the lipid bilayer, since D-bungarotoxin
binds to this domain. The inhibitory effect of Dbungarotoxin on
the ion-channel activity of AchR was not verified.
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