Tracking Single Proteins in Lipid Bilayers Using Fluorescence Microscopy

Proteins operate in a complex state space and much of it is hidden. We use single molecule imaging to reveal these states. When tracking single membrane proteins in a lipid bilayer, the main challenges are sample preparation, photobleaching, and time resolution.

To begin, remove the lipid stalk solutions from the freezer and allow them to reach room temperature. Take a clean 10 milliliter flask out of the oven and let it cool to room temperature. Add 900 microliters of spectroscopic grade chloroform, and 100 microliters of spectroscopic grade methanol to the cooled round bottom flask.

Then add the appropriate amounts of each lipid to the flask and swirl to mix thoroughly. Now place a vacuum distillation connector on top of the round bottom flask and attach it to the nitrogen drying line. Then turn on the nitrogen gas and adjust the pressure until the flow is just barely felt on the back of the hand.

Pipette one milliliter of buffer for every five micromoles of total lipid into the round bottom flask. Place a glass stopper on the flask and seal it with paraffin film. Position the flask and a clamp on a ring stand and submerge its bottom in a 60 degree Celsius sonicater bath.

Confirm that the solution becomes opaque and that no lipids remain attached to the inner wall of the flask. After the one hour incubation, turn on the sonicater and set the amplitude to its highest level. Move the flask around within the bath to locate the spot with the strongest agitation where the solution visibly bumps and sprays inside the flask.

Sonicate the solution for 30 minutes. Observing the transition from opaque to transparent and slightly opalescent. Remove the lipid solution from the flask and transfer it to a micro centrifuge tube.

Centrifuge the tube at 100, 000 G for one hour at four degrees Celsius. After centrifugation, remove and transfer the supernatant to a new centrifuge tube. Place the 25 millimeter quartz cover slips into a beaker and add equal volumes of nano pure water, 30%hydrogen peroxide and concentrated nitric acid.

Heat the cover slips in the prepared solution for 30 minutes until bubbling begins. Check the solution every 10 minutes and swirl gently to prevent the cover slips from sticking together. Observe the cover slips sliding apart and separating during swirling.

Once separated, gradually reduce the swirling speed to maintain their separation and allow the bubbling solution to coat the cover slips evenly. Then rinse the cover slips thoroughly with purified water while gently swirling to remove all chemical residue. Alternatively, clean the quartz cover slips using n-hexane followed by methanol, wiping with lens tissue for each solvent.

Place the cover slips inside a UV ozone cleaner with the surface to be treated facing the lamp. Allow oxygen to flow into the chamber at five pounds per square inch for five minutes. Then turn off the oxygen flow.

Now turn on the ultraviolet lights for 15 minutes, then allow the cover slips to rest for at least 10 minutes to permit the ozone to dissipate. Place a freshly cleaned cover slip into a 25 millimeter sample holder. Using a one quarter inch SM one lens tube, cut an eight millimeter diameter double layered par film gasket and position it in the center of the sample holder.

Apply a 50 microliter droplet of the small Unilamellar vesicles to the center of the eight millimeter gasket. After sealing the chamber, incubate at 37 degrees Celsius for one hour. Post incubation, use a pipette to rinse away the solution.

Add 50 microliters of fresh buffer to the bilayer and repeat this process a total of 10 times. After the final rinse, remove the buffer from the sample holder. Add a 50 microliter aliquot of the desired protein in detergent, ensuring the concentration is at or below the critical micella concentration.

Incubate the sample at 37 degrees Celsius for at least one hour to allow protein incorporation, rinse the sample with buffer to remove any unincorporated protein and detergent. Set the vertical pixel shift speed to approximately 600 nanoseconds and increase the vertical clock voltage using overclock mode. Then configure the horizontal pixel readout to its maximum speed.

Adjust the pre amplifier gain to two. Set the amplifier output for electron multiplication and set the electron multiplier gain to its highest level. Then set the exposure time to 25 miliseconds.

Confirm that the actual frame rate exceeds the exposure time slightly. Now adjust the laser power until the signal clearly stands out from the background noise. Collect enough imaging data to yield a minimum of 1000 tracks per sample.

For tracking analysis, crop all data sets to a consistent size and perform background correction using Image J.In Fiji, drag and drop the movie or stack TIFF file that needs to be analyzed into the interface. To enter the pixel calibration details, go to the analyze tab, select set scale, and apply the pixel to distance calibration specific to the instrument. Then save a copy of the dataset to preserve the original and track any changes made.

Subtract the background from a selected region of interest and apply it to the saved copy of the original data. Go to the process tab and choose subtract background. In the plugins tab, select tracking, followed by trackmate to launch the trackmate dialogue window for tracking analysis.

The degree of labeling for AQP four tetramers was calculated to be 4.12 using UV visible spectrometry and the Poisson distribution analysis indicated that 98%of Tetramers carried at least one fluorescent dye molecule. The histogram shows a wide distribution of step sizes consistent with diffusion of differently sized orthogonal arrays of particles, and the calculated average diffusion coefficient was 0.0143 square micrometers per second. In this video, we have identified key states important to aquaporin four regulation and their associated thermodynamic forces.

This protocol eliminates guesswork in creating biomimetic membranes with purified transmembrane proteins that are suitable for time lapse single molecule tracking. Protein machines operated at inflection points time lapse single protein tracking enables direct observation of stochastic pathways, branches, and dead ends.