A two-step approach was used to incorporate the connector into the planar bilayer lipid membrane (BLM). The first step was the preparation of unilamellar lipid vesicles containing the reengineered connector as described above. The next step was to fuse the extruded liposome into a planar BLM (Fig. 2i ). The fluidity of the lipid bilayer was demonstrated by FRAP (Fluorescence Recovery After Photobleaching) (Fig. 2h ). An excitation light was focused continuously on the bilayer to bleach the dye. The photobleached area appeared dark. But after the light was off, it gradually recovered due to the diffusion of the fluorescent lipid.
A standard BLM chamber (BCH-1A from Eastern Sci LLC) was utilized to form horizontal BLMs. A thin Teflon film with an aperture of 70–120 µm (TP-01 from Easter Sci LLC) or 180–250 µm (TP-02 from Easter Sci LLC) in diameter was used as a partition to separate the chamber into cis- (working volume 250 µL) and trans- (working volume 2.5 mL) compartments. After the aperture was pre-painted with 0.5 µL 3% (w/v) DPhPC n-decane solution twice to ensure the complete coating of the entire edge of the aperture, these compartments were filled with conducting buffers (5 mM Tris/pH 7.9, TMS, or 5 mM HEPES/pH 7.9, with varying concentration of NaCl or KCl).
Formation of the bilayer membrane on the partition is a key step for connector insertion into the bilayer (Fig. 2i ). Considering all experiments, the occurrence of successful connector insertions was about 47–83%, which varied from person to person based on BLM experience and the quality of prepared proteoliposomes. So far, we have carried out a total of 280 separate BLM experiments in which successful connector insertions were found.
For single conductance measurements, the giant liposome/connector complex prepared earlier must be extruded using a polycarbonate membrane with pore size of 200 nm or 400 nm to generate small unilamellar liposomes. This liposome stock solution was further diluted by 10–20 fold for the BLM experiments before use. For insertion of connectors, 0.5–2 µL of the diluted liposome solution was loaded into the cis-chamber.
Conductance was measured in two ways: the first was derived at specific but constant holding potentials, and the second from the slope of the current trace induced by a scanning potential starting at −100 mV and ramping to 100 mV after incorporation of GP10 connector into the lipid membrane (Fig. 3f and 3g ).
A standard BLM chamber (BCH-1A from Eastern Sci LLC) was utilized to form horizontal BLMs. A thin Teflon film with an aperture of 70–120 µm (TP-01 from Easter Sci LLC) or 180–250 µm (TP-02 from Easter Sci LLC) in diameter was used as a partition to separate the chamber into cis- (working volume 250 µL) and trans- (working volume 2.5 mL) compartments. After the aperture was pre-painted with 0.5 µL 3% (w/v) DPhPC n-decane solution twice to ensure the complete coating of the entire edge of the aperture, these compartments were filled with conducting buffers (5 mM Tris/pH 7.9, TMS, or 5 mM HEPES/pH 7.9, with varying concentration of NaCl or KCl).
Formation of the bilayer membrane on the partition is a key step for connector insertion into the bilayer (
For single conductance measurements, the giant liposome/connector complex prepared earlier must be extruded using a polycarbonate membrane with pore size of 200 nm or 400 nm to generate small unilamellar liposomes. This liposome stock solution was further diluted by 10–20 fold for the BLM experiments before use. For insertion of connectors, 0.5–2 µL of the diluted liposome solution was loaded into the cis-chamber.
Conductance was measured in two ways: the first was derived at specific but constant holding potentials, and the second from the slope of the current trace induced by a scanning potential starting at −100 mV and ramping to 100 mV after incorporation of GP10 connector into the lipid membrane (
