The overall goal of our easy to implement procedure is to demonstrate fast detergent-free delivery of membrane proteins into lipid bilayers of various sizes using fusion between lipid bilayers formed of oppositely charged lipids. Our methodology can be used in biochemistry and biophysics for one step assembly of biomer metric multi-component lipid bilayer systems from layer individual components. It is important to mention that the advantage of our approach is in this fast functionalizition, or namely complex model lipid bilase, with proteins, lipids and other molecules of interest.
This methodology can also be used to study membrane transport processes that require energization of the lipid membrane. For example, primary pumps can be used to drive secondary transport enabled by other membrane proteins. Our method can also serve in needs of synthetic biology.
For example, to create membrane vesicles with various program functionalities. In future, trapping small vesicles like these inside similar larger ones, maybe a step toward construction of an artificial cell. To form the lipid and oil solution, mix Chloroform stock with one milliliter of hexadecane in a 1.5 milliliter micro centrifuge tube.
Next, to evaporate the Chloroform, constantly mix and heat the mixture in a horizontal shaker at 80 degrees Celsius for 30 minutes by leaving the tube open. After evaporation, close the tube and cool down to room temperature. Next, to form the lipid monolayer, at the lipid in oil aqueous buffer interface, take a 1.5 milliliter centrifuge tube, and transfer 200 microliters of lipid in oil solution on top of 0.5 milliliters of Buffer B.Then wait for 30-60 minutes at room temperature for the phase separation border to flatten, indicating the formation of the lipid monolayer.
The convex shape of the phase separation border is due to surface tension mismatch between the oil and the aqueous buffer. Next, to prepare the water and oil emulsion, mix a solution of non-ionic poly saccharide in Buffer B with a density high than that of water. Then transfer 0.5 microliters of the mixture to a new 1.5 milliliter centrifuge tube, containing 100 microliters of lipid in oil solution.
Then sonicate the mixture at 44 kilohertz and 14 watts for 30 seconds in an ultra sonic water bath. After sonicating, vigorously vortex for 45 minutes to form the water in oil emulsion. Then, to convert the water in oil emulsion into anionic GUV carefully transfer the emulsion on top of lipid and oil aqueous interface.
In the emulsion, each droplet is coated with lipid monolayer. Proper syndication vortexing of the water in oil emulsion determines success of GUV formation. If not formed correctly, the emulsion droplet will coalesce in turn, reducing the yield of GUV formation.
To obtain the GUV pellet, quickly centrifuge the sample at 1000 times G for two minutes. Place a bent metal wire in the tube so that it sits within the oil. Then leave the tube on ice to solidify the lipid and oil mixture.
Carefully remove the frozen oil and collect the aqueous phase. Next, re-suspend the GUV pellet in 50 microliters of fresh Buffer B and transfer to a new tube. Inspect the GUV under a 100 X oil emersion objective of a fluorescent microscope.
Use gel filtration resin to prepare a gravity flow column and equivalate with Buffer A kept at room temperature. Then dilute the purified membrane protein to a final concentration of 0.7 milligrams per milliliter using the extraction buffer. Then, mix the protein with pre-formed cationic SUV in a one to 30 ratio along with collate containing Buffer A to a final volume of 660 microliters.
Then, place the sample tube on a rocker and gently shake the mixture for 15 minutes. Next, pass the mixture through gel filtration resin and collect the turbid proteoliposome fractions and centrifuge at 4, 000 times G for 15 minutes. Then, discard the non-reconstituted protein supernatant and dissolve the pellet in one milliliter of fresh Buffer A.In a fluorometer cuvette, add 20 microliters of proteoliposome with B03 oxidase along with two milliliters of Buffer A in the presence of 0.5 micromolar ACMA, and wait for stable signal at 430 nanometers excitation, and 515 nanometers emission.
Then, add 40 micromolar of coenzyme Q1 to the cuvette. Next, add two millimolar DTT to the proteoliposome to start proton pumping, and then dissipate the formed delta pH. In a separate cuvette, add 40 microliters of proteoliposomes with F1-F0 to 2 milliliters of Buffer A in the presence of 0.5 micromolar ACMA and trigger proton pumping by adding 0.2 millimolar ATP to the proteoliposome and then dissipate the formed delta pH.
Fuse 50 microliters of PL+with 50 microliters of 800 nanometers LUV-in one milliliter of Buffer D, supplemented with one millimolar of Magnesium Chloride, and 20 millimolar Potassium Chloride for five minutes. Then centrifuge the sample at 6, 000 times G for five minutes to pellet the post fusion LUV. After centrifugation, discard the supernatant consisting of unreacted PL+then resuspend the pellet formed in one milliliter of Buffer D, supplemented with one millimolar of Magnesium Chloride and 100 millimolar Potassium Chloride.
Again, conduct the ACM Quenching Assay. First, mix together three microliters of B03 oxidase PL+and five microliters of F1F0 PL+then fuse the mixture with three microliters of LUV-or GUV-in 800 microliters in Buffer D, supplemented with 20 millimolar Potassium Chloride and one millimolar Magnesium Chloride for five minutes. Next, add Potassium Chloride and Mops Buffer to the fused membrane mixture at a final concentration of 150 millimolar.
Mix 400 micromolar ADP, 50 micromolar luciferin and 2.5 of luciferase in 200 microliters of Buffer A to prepare a luciferin luciferase ADP cocktail. Then, add the cocktail to the fused membrane mixture. For correct ADP synthesis measurement, it is important to have a stable luciferase signal to avoid false positive, before addition of Q1 and DTT.
Then add 40 micromolar oxidized coenzyme Q1, and then add two millimolar DTT to initiate the energization of the fused membrane. After one minute, add a five millimolar Potassium Phosphate solution to the energized vesicles to start ATP synthesis. To inspect the resuspended GUV on removing the frozen hexadecane from the lipid and oil mixture, fluorescent microscopy was utilized.
Images of the GUV obtained show the presence of fluorescent cholesterol biopoty FL12 on its membrane, and self erodimine 101, a polar fluorophore in the lumen. To quantify the fusion of SUV in the increasing concentration of Potassium Chloride, a graph was plotted. The graph explains the with increasing Potassium Chloride concentration beyond 50 millimolar, the fusion of the SUV lipid vesicles starts decreasing.
However, at low Potassium Chloride concentration, between one to 20 millimolar, the complementary charged vesicles fuse. Next, to measure the BO3 oxidase meditated proton pumping drive oxidation of coenzyme Q1 substrate, a graph was plotted based on ACMA Quenching Assay. The results show that adding an uncoupler increase the ACMA Fluorescence signal represented along the Y axis of the plot.
Then, ATP hydrolysis dependent proton pumping by F1F0 was also measured based on ACMA Quenching Assay. In the graph, it shows that adding an uncoupler increases the AMCA Fluorescence signal. Further, luciferin luciferase system was used to monitor ATP synthesis in fused vesicles.
Sequential addition of coenzyme Q1 DTT, followed by Phosphate addition, shows successful ATP production by the fused vesicles. Once mastered, the first technique formation of fusogentic proteoliposome from detergents symbolized membrane proteins and union myomedicals can be done in just 30 minutes. The second technique, physical fusion, can be done even faster within just ten minutes.
After watching this video, you should have a good understanding of how one could construct multi-component membrane systems by combining various individual building blocks that are presented by functionalized lipid vesicles. We expect such modular systems where the chief properties and functions that are impossible with one step re-constitution of membrane proteins. In general, our procedures provide a simple framework for building complex compartmentalized bio-chemical systems, from simpler, so-called bio-bricks.
