A Fluorescence-based Assay of Phospholipid Scramblase Activity

The overall goal of this procedure is to reconstitute a typical G protein coupled receptor into large unilamellar vesicles, and to measure it's ability to carry out 80p independent translocation of phospholipids across a lipid bilayer using a flurorescence-based assay. This method can help answer key questions in the inter-cellular lipid transport field, such as what is the molecular identity of proteins that can scramble off liplipids and what is their mechanism. The main advantage of this technique is that it is very reliable and robust.

Once your reconstitution procedure is established for a given lipid composition and a preferred detergent. To begin this procedure, use a glass syringe to add 1435 microliters of a 25 milligram per milliliter solution of POPC and chloroform to a round bottom flask. Add 160 microliters of a 25 milligram per milliliter solution of POPG and chloroform to obtain 52.5 micro-malipids in a nine to one molar ratio of POPC to POPG.

Next, dry the lipids for 30 minutes using a rotary evaporator set to 145 RPM. Once drying is complete, transfer the flask to a vacuum desiccator for three hours at room temperature. Then, add 10 milliliters of buffer A to hydrate the died lipid film.

Gently swirl the flask until a homogeneous turbid suspension is formed. Sonicate the suspension at a frequency of 40 kilohertz in a room temperature water bath for 10 minutes. Then, using an extruder, pass the sample through a membrane with a 400 nanometer pore size, 10 times.

Follow this with a second cycle of extrusion, passing the sample through a membrane with a 200 nanometer pore size, four times. Set aside the resulting visibly clearer suspension, which contains large unilamellar vesicles of phospolipids about 175 nanometers in diameter. After the extrusion is complete, prepare standards in 13 by 100 millimeter glass tubes as outlined in the text protocol.

Then, add 10 microliters of each LUV and proteoliposome sample to a unique glass tube and dilute with 40 microliters of deionized distilled water. Add 300 microliters of perchloric acid to each tube, and heat for one hour at 145 degrees Celsius in a heating block. Place a marble on each tube to prevent evaporation.

Then let the tubes cool to room temperature and add one milliliter of deionized distilled water to each. Add 400 microliters of a freshly prepared solution containing 12 grams per liter of ammonium molybdate and 50 grams per liter of sodium ascorbate to each, and vortex to mix. Heat the tubes for 10 minutes at 100 degrees Celsius using marbles to prevent evaporation.

Then remove the tubes from the heating block and allow them to cool to room temperature. Using a spectrometer set to 797 nanometers, obtain a calibration curve using the standards. Then, measure the absorbence of the samples against the blank, and determine the phosphate content.

Begin by pipetting 800 microliters of each extruded LUV suspension into a unique two milliliter microfuse tube. Add 5.3 microliters of buffer A and 34.7 microliters of 10%mass-per-volume DDM, dissolved in buffer A, to each tube. Next, incubate the samples for three hours at room temperature with end-over-end mixing.

While the samples are incubating, weigh out 400 milligrams per sample of beads in a glass beaker. Wash the beads with five milliliters per sample of methanol, stirring slowly for 10 minutes. Repeat this wash once for methanol, three times with water, and once with buffer A.During the last 30 minutes of sample incubation, dry 9.5 microliters-per-sample of NBD labeled phospholipid in a screw-cap glass tube under a stream of nitrogen.

Then, add 45 microliters-per-sample of 0.1%mass-per-volume DDM in buffer A to dissolve the dried phospholipid. After incubation is complete, add the dissolved MBD labeled phospholipid solution to each sample. Then, add 0.1%DDM, buffer A, and the DDM-solubilized opsin in the desired amount, in that order, to obtain a final volume of one milliliter with a concentration of seven millimolar DDM.

Reconstitution of a membrane protein is achieved by treating vesicles with sufficient detergent so that the swell will not dissolve. And that these conditions, which have to be empirically found for every lipid composition and detergent, the protein will integrate into the liposomes. Mix the samples for one hour at room temperature.

Then, add 80 milligrams of the prepared polystyrene beads to each tube, and incubate again for one hour at room temperature with end-over-end mixing. Add an additional 160 milligrams of polystyrene beads to each sample, and incubate for two hours at room temperature with end-over-end mixing. When the incubation is complete, transfer the samples, leaving the beads behind, to clean, glass tubes, each containing 160 milligrams of fresh beads.

Mix end-over-end overnight at four degrees Celsius, protected from light. Next, transfer the samples, without the beads, to unique microfuse tubes. Place the tubes on ice in preparation for the scramblase activity assay.

Begin by adding 1950 microliters of buffer A to a plastic cubette, containing a plastic stir bar. Then, add 50 microliters of the prepared protealiposome sample. Let the sample equilibrate in the fluorescent spectrometer with constant stirring for five seconds.

While the sample is equilibrating, prepare a solution of one molar dithionite in 0.5 molar unbuffered tris. Start the fluorescence monitoring with an excitation of 470 nanometers, an emission of 530 nanometers, and a slit width of 0.5 nanometers. Record for 50 seconds to achieve a stable signal.

Then, add 40 microliters of one molar dithionite solution to the cubette. Record the fluorescence for at least 500 seconds. Finally, analyze the data as outlined in the text protocol.

For a complete analysis of the fluorescence data, when it's to determine the fraction of vesicles that cannot be reconstituted. The recovery of proteins and lipids in the reconstituted vesicles and also the size distribution of the vesicles. In this study, opsin is reconstituted into large unilamellar vesicles to characterize its scramblase activity using a fluorescence-based assay.

Vesicles are reconstituted with NBD-PC, which equally distributes between the outer and inner leaflets. Dithionite reduces the nitro group of NBD on the outside of the vesicle to a non-fluorescent amino groups resulting in a 50%reduction of fluorescence in protein-free liposomes. However, when opsin facilitates NBD movement between the inner and outer leaflet, a complete loss of fluorescence occurs.

The representative results of this approach at different protein-to-phospholipid ratios can be seen here. The extend of fluorescence reduction increases with the amount of opsin reconstituted into vesicles, which ranges from zero to 4.95 micrograms. Dithionite was added at the indicated time, and NBD fluorescence was monitored for 400 seconds.

Fluorescence reduction is more significant at higher protein-to-phospholipid ratios, with the largest reduction being 85%The endpoint fluorescence reduction is then used to determine the probability of scramblase activity, the likelihood of vesicles having at least one scramblase using Polson statistics. The red curve represents a mono-exponential fit for opsin. While the dashed grey lines represent mono-exponential fits for the reconstitution of opsin into vesicles as monomers, pre-formed dimers, or pre-formed tetramers.

The method we describe can easily be adapted to different needs. The procedures are versatile, and can be used to identify and characterize phospholipid scramblase activity of other membrane proteins in the future. The reconstitution procedure can be applied to any membrane protein.

This technique enabled us to identify opsin as the first biochemically verified phospholipid scramblase and allowed us, then, to characterize the activity of different confirmation states of the protein, and its ability to transport different lipids.