Single-molecule FRET can capture dynamics happening at nanometer distances, which is the right scale for ribosomal protein biosynthesis process. Ribosome works by coordinating multiple factors and components allosterically, which is intrinsically inhomogeneous. Single-molecule method can track each ribosome without being limited by this inhomogeneous average effect.
This method will reveal how antibiotics inhibit ribosome function to develop new drugs toward drug-resistant bacterial infections. Begin by preparing the PRE by mixing the initiation EFTU, NOG, and PAG mix at the ratio of one-to-two-to-two at 37 degrees Celsius for two minutes. After incubation, purify the PRE using 1.1 molar sucrose cushion ultracentrifugation overnight at 100, 000 times G.Prepare the POST by mixing the initiation EFTU, WG, and PHE mix at a ratio of one-to-two-to two at 37 degrees Celsius for 10 minutes.
After incubation, purify the POST using 1.1 molar sucrose cushion ultracentrifugation overnight at 100, 000 times G.Clean the microscope glass slides containing six pairs of holes that are one millimeter in diameter and the number 1.5 microscope glass coverslips. Bake the clean slides and coverslips at 300 degrees Celsius for three hours, then coat the coverslip with aminosaline. In a clean laminar hood, lay one coverslip flat on the surface.
Carefully drop 60 microliters of PEG solution on the top edge, then lay another coverslip on top of it, letting the capillary effect spread the solution between them ensuring that no bubbles are formed. Repeat these steps for two more pairs of coverslips. Store these coverslips in an empty tip box filled with water and incubate them for three hours in the dark.
After three hours, separate the coverslips, rinse with water in three consecutive crucibles and purge with a dry nitrogen stream. Pull sharp pipette tips through the holes on the glass slides until they fit tightly. Scrape the sharp ends off until they are completely flat with the glass surface, then cut a double-faced tape with the channel pattern on it.
Stick the onto the glass slides on the flat side. Stick the coated coverslip onto the double-face tape and press on it to make a tight seal of the sample chamber, making sure the coated side is facing inward. Turn on the computer and the microscope switch.
Start the laser control interface and turn on the laser. Push the enable button on the laser control box to warm up the laser. Turn on the cameras, then start the microscope-associated software program.
Click the upper shutter off and click the lamp on. Prepare the TAM10 with Tween buffer by adding 10 microliters of 5%Tween-20 into one milliliter of TAM10 buffer. Prepare the deoxy solution by weighing three milligrams of glucose oxidase in a small microcentrifuge tube and add 45 microliters of catalase to it.
Vortex the tube gently to dissolve the solid. Spin at 20, 000 times G in a microcentrifuge for one minute and take the supernatant. Prepare 30%glucose solution, 200 millimolar Trolox solution and 0.5 milligrams per milliliter of streptavidin solution following instructions in the text manuscript.
Add one drop of imaging oil to the turf objective. Put the sample chambers on the objective. Fill the chamber with 10 microliters of streptavidin solution and wait for one minute.
Wash the chamber with 30 microliters of TAM10 with Tween buffer, collecting the run-through solution with a folded filter paper. Wait for one minute after washing. Dilute the ribosome complexes PRE or POST to 10-15 nanomolar concentrations with TAM10 with Tween buffer.
Load 20 microliters of the ribosome sample into the channel and wait for two minutes. Make the imaging buffer using 50 microliters of TAM10 buffer and 0.5 microliters each of deoxyglucose and Trolox solution. Mix them well.
Flush the chamber with 30 milliliters of the imaging buffer. Set the camera for acquiring images. Click the upper shutter on and the lamp off.
Start the camera acquisition and spread the images into three windows representing two individual cameras and the overlay. Under the acquire menu, select capture timelapse, and then click on capture automatically. Set the appropriate file name, file path, and the number of loops and click on run now.
Close the pop-up window once the image acquisition is complete and then open the acquired ND file. Click on ROI, simple ROI editor and choose the option circle. Select the ROI on the image and click on finish when it is completed.
Click on measurement, time measurement to show the data either as a plot or a spreadsheet. Open the export tab to set the export parameters, then click on export to save the intensities. Finally, open the file which has been exported using a suitable application.
The distance from the L27 labeling residue to the A-site or P-site tRNA is 61 or 52 angstrom respectively, corresponding to FRET efficiency of 0.47 and 0.65. Florescence intensities from the donor and acceptor channels were retrieved and plotted as timelapses. After donor bleaching, both traces approached the baseline because no excitation could occur directly on the acceptor.
After acceptor bleaching, the donor intensity increased because fewer reaction pathways dissipated the excitation energy. The individual ribosomes exhibited different fluctuations due to the wobbling motion of the tRNAs, which caused distance fluctuations to the L27. Other methods like Puromycin assay, assay, toeprinting assay and mass spec are complimentary to single-molecule FRET method and reveal more details about protein synthesis.
Single-molecule FRET has a broad application because many biological processes happen in the nanometer range and at millisecond timescales. By tagging proper dyes at proper positions, many other dynamics can be revealed.
