Here, we used single-molecule FRET, and site-specific protein labeling via a natural amino acid incorporation to study the conformational dynamics of mGluR2. This allows to study the protein dynamics without perturbing protein structure. Atomic structure provide a static image of a protein.
However, single-molecule FRET enables us to visualize the full range of protein motion in real-time and solution. This method can be applied to study the dynamics of any protein. Moreover, a natural amino acid incorporation can be used to study protein-protein interactions, and the engineering of synthetic receptors.
Sample loading and optimization of imaging requires patience and practice. To begin, mark holes to be drilled on the glass slide with a marker. Use a Dremel to drill small holes on the slide while the slide is dipped in water.
Sonicate the slides and cover slips in acetone for 30 minutes in a bath sonicator at 23 degrees Celsius. Dispose of acetone from the slide and cover slip jars. Rinse thoroughly with water, and sonicate in five-molar potassium hydroxide for 30 minutes.
Next, rinse the slides and cover slip with water, and sonicate in methanol for two minutes. Leave the jars filled with methanol until the amino salinization step. Pour freshly prepared amino salinization mixture into the slide and cover slip jars.
Incubate, sonicate, and again incubate the slides and cover slips in an amino salinization mixture before discarding the solution. Next, wash the jars with methanol before filling them with water. Then rinse the slides with water.
Dry them with air blow, and place them in the assembly boxes. Apply 60 milliliters of PEGylation solution to the slide, and place a cover slip on top of it. Mark the non-PEGylated side before storage.
After incubation, gently disassemble, and rinse the slides and cover slips with water. Then dry them by blowing air, and place them in a sterile 50-milliliter tube with the PEGylated surface facing away from each other. After passaging HEK 293T cells with 0.05%trypsin-EDTA, seed the cells on poly-L/D-lysine cover slips.
Place the standard media with AZP supplemented media for growing cells and metabotropic glutamate receptor 2, or mGluR2 expression. Then transfect the cells with a transfection reagent, and incubate for 24 hours before changing the medium with fresh AZP supplemented media. 20 minutes before labeling with alkyne cyanine dyes, wash the cover slips twice with warm Recording Buffer, or RB, and place them in a warm standard media with no AZP.
Then remove the standard media and wash the cells with RB before adding the freshly prepared labeling solution. Incubate for 15 minutes at 37 degrees Celsius in the dark. After incubation, remove the labeling solution.
Wash the cover slip containing transfected cells twice with the RB, and re-suspend in the same medium. Next, pellet the cells by spinning at 1, 000 g at four degrees Celsius for five minutes, and remove the supernatant before re-suspending the pellet in 80 to 130 microliters of the lysis solution. Break the pellet by pipetting.
Then wrap it in foil, and place it on the rocker at four degrees Celsius for 30 minutes to one hour to lyse the cells. Pellet the insoluble fraction by centrifugation at 20, 000 g and four degree Celsius for 20 minutes. Then transfer the supernatant to a fresh cold tube, and store it on ice.
Remove the slide and cover slip from the freezer, and allow them to warm at room temperature in the dark. Assemble the chamber by sandwiching the strips of double-sided tape between the slide and cover slip, ensuring the PEGylated surfaces form the interior of the flow chamber. Using a pipette tip, press the cover slip to ensure the tape is contacting the cover slip and slide.
Apply epoxy to the edges of the slide. Apply 40 microliters of 500-nanomolar NeutrAvidin to each lane, and incubate inside a humidified dark box. After two minutes, wash each chamber lane with 100 microliters of T50 buffer.
Then add 20 nanomolar biotinylated antibody solution, and incubate for 30 minutes before washing with 200 microliters of T50 buffer per lane. Turn on the computer and microscope. Then turn on the lasers to warm up.
Next, turn on the EMCCD camera, and open the software. Mount the sample chamber on the microscope stage, and add the protein sample gradually to achieve approximately 400 molecules per field of view. Wash the chamber with 100 microliters of imaging buffer supplemented with 0.3 units of protocatechuate 3, 4-dioxygenase.
Adjust the gain, acquisition rate, and laser powers to detect single-molecule fluorescence signals in both the donor and accepter channels. Then excite the donor, and acquire time traces until 80%of the donor molecules in the field of view are photobleached. At the end of the movie, turn on the 640-nanometer laser to directly excite the accepter until some of the molecules are photobleached.
Change the field of view, and repeat the steps for excitation of donor and accepter to collect three movies per condition. For selecting single-molecule FRET traces of particle picking, select the traces where the total intensity of donor and accepter traces is stable over time. Then select the traces with anti-correlated changes in donor and accepter intensities.
Select the donor and accepter molecules showing single-step photobleaching and traces more than five seconds long. Calculate the FRET deficiency. Finally, identify the conformational state by Hidden Markov Modeling, or HMM, by following the steps described in the manuscript.
The labeling of AZP by cyanine dyes was achieved by a copper-catalyzed cycloaddition reaction, and resulted in effective plasma membrane labeling of 548UAA with the cell surface population labeled with donor and accepter fluorophores. In SDS-PAGE electrophoresis, a single band at 250 kilodalton was observed in HEK 293T cells expressing 548UAA, which coincided with the full-length dimeric mGluR2. Representative selective traces for multiple short-lived and long-lived for donor and accepter bleaching events are shown here.
Conformational states were identified using HMM analysis. Transitions between discrete FRET states were extracted from the idealized fits, and plotted as a transition density heat map. The idealized FRET traces also yield information on dwell time for each identified confirmation.
The single-molecule's traces with donor and accepter channel is shown here. Further, single molecule FRET reveals four conformational states of the mGluR2 cysteine-rich domain. Efficient peak preservation is essential to single-molecule pull-down, and care should be taken during this process.
In addition, the oxygen-scavenging agent should be added immediately prior to imaging. This labeling method and single-molecule FRET experiments have been applied to multiple receptors, and have provided new insights into the mechanism of receptor activation and modulation of receptor function.
