We are committed to study the fusion mechanism of intracellular membranes. In this protocol, we aim to realize the conformational dynamics of the endoplasmic reticulum membrane fusion protein atlastin during its GTP hydrolysis cycle by smFRET. Atlastin may exist as a monomer or dimer in the GTP hydrolysis cycle.
In single-molecule experiments, it's challenging to find appropriate protein labeling and immobilization strategies, and the following presents the conformational dynamics of atlastin throughout the GTP hydrolysis cycle. Compared with bulk FRET, smFRET can accurately monitor protein conformations under different nucleotide-loading states, thus providing direct insight into the behavior of modulator molecules. We used the smFRET to fully resolve GTP hydrolysis cycle of atlastin during different position strategies.
We believe that our findings will promote more conformational dynamics in studying GTP hydrolysis proteins, and provide new interpretations of rare biological process. To clean the surface of the coverslip, use tweezers to pick up eight coverslips, and place them in a staining jar. Add 50 milliliters of acetone to cover the coverslips, and place the staining jar in an ultrasonic cleaner for 30 minutes.
Rinse the coverslips three times with double-distilled water, and wash them with methanol in an ultrasonic cleaner. Next, add piranha solution to the staining jar, and heat it in a water bath kettle at 95 degrees Celsius for two hours. After cooling the jar to room temperature, rinse the coverslips six times with double-distilled water.
Add sodium methoxide solution into the staining jar, and place it in the ultrasonic cleaner for 15 minutes. After rinsing the coverslips with water, add double-distilled water to the staining jar, and place it in the ultrasonic cleaner for 15 minutes. Clamp the coverslips in the staining jar and dry them with nitrogen.
Transfer the dried coverslips into another staining jar, and bake the jar in a drying oven at 120 degrees Celsius for 30 minutes. Then, cool the jar to room temperature in a desiccator. Add 47.5 milliliters of methanol, 2.5 milliliters of acetic acid, and 0.5 milliliters of triethoxysilane to a beaker and mix evenly.
Transfer the mixture to the staining jar and incubate for 10 minutes. Rinse the coverslips three times with double-distilled water in the staining jar, and sonicate for five minutes. After rinsing the coverslips with water and drying them with nitrogen as demonstrated, place the coverslips into a 10-centimeter diameter Petri dish.
Dissolve SVA-mPEG and SVA-mPEG-Biotin in the high-salt solution at a ratio of 1:100. Drop the prepared mix onto a coverslip and cover it with another coverslip. Incubate the coverslips with appropriate humidity for two hours or overnight.
After modification, separate the coverslips, rinse them with deionized water, and blow dry with nitrogen. Carefully place the modified coverslip in a 50-milliliter tube. After purifying the GTPase domain of dynamin-like protein atlastin or ATL expressed in Rosetta E.coli cells, change the protein buffer to the protein biotinylation buffer using a 10-kilodalton centrifugal filter.
For protein biotinylation, mix 100 microliters each of 100-millimolar ATP, 100-millimolar magnesium acetate, and 500-micromolar D-biotin, 10 microliters of 100-micromolar BirA biotin ligase, and 50-micromolar of protein to a final volume of one milliliter. Incubate the mixture overnight at four degrees Celsius. The next day, remove free D-biotin using a 10-kilodalton molecular weight cut-off centrifugal filter.
Incubate 20 microliters of biotinylated protein with 20 microliters of 15-micromolar streptavidin for 20 minutes on ice. Detect the efficiency of protein biotinylation using SDS-PAGE. Dissolve LD555 and LD655 fluorophores in dimethyl sulfoxide to a final concentration of five millimolar.
To prepare the labeling buffer, mix 25 millimolar HEPES, 150 millimolar potassium chloride, and five millimolar magnesium chloride. Change the protein buffer to the labeling buffer using a 10-kilodalton centrifugal filter. For intramolecular smFRET assays, mix ATL1cyto-TK with LD555 and LD655 at a ratio of 1:1.2:1.2 to a final volume of 100 microliters.
Incubate the mixture for five hours at four degrees Celsius. For intermolecular smFRET assays, incubate ATL1cyto-K with LD555 and biotinylated ATL1cyto-K with LD655 for five hours at four degrees Celsius. To prepare the protein immobilization chamber, carefully stick the modified coverslip and microscope slide with customized double-sided tape.
Install hoses and tips to form a microfluidic chamber with six channels. For mapping corrections, add 10 microliters of 10%polystyrene particles onto a microscope slide and cover it with a coverslip. Then, under a total internal reflection fluorescence microscope, select a field of view containing polystyrene particles and capture a video in a bright-field mode.
Use a custom script to align the center location of the same polystyrene particle in both the donor and acceptor channels. Save the map file as a TXT file. For intermolecular smFRET experiments, mix LD555 labeled ATL1cyto-K with LD655 labeled ATL1cyto-K-biotin at a one-to-one ratio with a final concentration of one millimolar of GTP-gamma-S.
Incubate the mixture on ice for one hour to dimerize the proteins. Mix LD555 labeled ATL1cyto-T with LD655 labeled ATL1cyto-K-biotin at a one-to-one ratio with one millimolar of GTP-gamma-S for ATL1cyto-T and ATL1cyto-K intermolecular smFRET experiments. Incubate the mixture on ice for one hour to obtain dimerized proteins.
Then, for intramolecular smFRET assays, incubate LD555 and LD655 labeled ATL1cyto-TK with one millimolar of GDP on ice for one hour. For intermolecular experiments, incubate 10 micrograms per milliliter of streptavidin for 10 minutes to immobilize proteins. For intramolecular assays, add 10 micrograms per milliliter of biotinylated anti-His antibody and incubate for 10 minutes to immobilize proteins.
After incubation, add the diluted ATL1 dimer to the protein in the channel and allow it to immobilize for 10 minutes. Flush the channel twice with an incubation buffer. Add benzoic acid and PCD to the channel at a final concentration of 2.5 millimolar.
Select the 532 nanometer laser as the excitation light source. Set the EMCCD camera to record data at a frame interval of 30 milliseconds. Save the recorded movies in 16-bit TIFF format.
After data acquisition, extract FRET tracks from the recorded movies. Select and save FRET tracks in TXT format. Extract the data from the TXT files, and calculate the FRET values using homemade scripts in MATLAB.
Fit the smFRET data by GaussAmp in Origin to obtain the distribution histogram.
