Biomedical Engineering Reference
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The insertion of the connector protein into the lipid membrane was confirmed by
fluorescence microscopy, filtration assay, and sedimentation analysis (Fig.
4.3
).
The presence of the fluorescently labeled connector in the membrane was demon-
strated by a clear fluorescent ring around the liposome (Fig.
4.3a-i
). The fluorescent
ring was very similar to the liposome generated with fluorescent lipids NBD-PE
(Fig.
4.3a
-ii). No fluorescent ring was observed when the fluorescently-tagged
connector was mixed non-specifically with the non-connector inserted liposome
(Fig.
4.3a
-iii). The free connectors were then removed by filtration using a 0.45
m
m
filter membrane or by 5-20% sucrose gradient ultracentrifugation (Fig.
4.3b, c
).
Furthermore, the lipid membrane retained its fluidity after incorporation of
the connector protein as demonstrated by FRAP (Fluorescence Recovery After
Photobleaching) (Fig.
4.3d
).
None of the aforementioned assays can distinguish between the membrane
surface attachment of the connector and incorporation into a membrane to form a
channel. A direct verification of the incorporation of a membrane channel is by
single channel conductance assay, described in the next section.
Fig. 4.3 (a) Epifluorescence Images of giant liposome (~50
m
m) containing the connector: (i)
lipid labeled with NBD-PE without connector; (ii) proteoliposomes reconstituted by FITC labeled
connectors; (iii) FITC-connector mixed non-specifically with liposomes. (b) Membrane filtration
isolated most of the free connectors. (c) Separation of liposome/FITC-connector complexes by
sucrose gradient sedimentation. Free connectors appeared in the top fractions while proteolipo-
somes remained in the lower fractions. Fractions #1-12 are not shown. (d)Fluidityoffluorescent
lipid bilayer demonstrated by FRAP (Fluorescence Recovery After Photobleaching) showing that
the fluorescence intensity of photobleached area (
black
) was gradually increased over time due to
lipid diffusion. (e) Schematic showing the insertion of the connector into a planar lipid bilayer via
vesicle fusion. Figures reproduced with permissions from: Ref. [
42
],
Nature Publishing Group
#
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