This protocol will help researchers more accurately determine the native oligomeric state of membrane proteins by utilizing the native cell membrane nanoparticle system in conjunction with electron microscopy. The advantage of this technique is that it provides accurate structural data of membrane proteins in a native cell membrane-like environment, and it can be utilized for future high-resolution structure determination. Demonstrating the procedure will be Kyle Kroeck, a postdoctoral fellow from my laboratory.
To prepare native cell membrane nanoparticles, resuspend one gram of the membrane pellet of interest in 10 milliliters of NCMN Buffer A.Use a glass Dounce homogenizer to homogenize the resuspended cell membrane sample at 20 degrees Celsius and transfer the suspended sample to a 50 milliliter polypropylene tube. Add membrane active polymer stock solution and additional NCMN Buffer A to the sample to a final concentration of 2.5%membrane active polymer and shake the solution for two hours at 20 degrees Celsius. At the end of the incubation, ultracentrifuge the sample.
During the centrifugation, equilibrate a five milliliter nickel NTA column with 25 milliliters of NCMN Buffer A.At the end of the centrifugation, transfer the supernatant onto the column and use a syringe pump set to a 0.5 milliliter per minute flow rate to load the supernatant onto the column at 20 degrees Celsius. Wash the fast protein liquid chromatography machine lines with enough NCMN Buffer B to completely flush the system and connect the column to the chromatography machine. Wash the column with 30 milliliters of NCMN Buffer B at a one milliliter per minute flow rate and collect the flowthrough.
Wash the column with 30 milliliters of NCMN Buffer C at a one milliliter per minute flow rate and collect the flowthrough. Elute the protein with 20 milliliters of NCMN Buffer D at a 0.5 milliliter per minute flow rate and use a fraction collector to collect one milliliter fractions of the sample, then store the protein samples at four degrees Celsius using SDS-PAGE to check the samples that correspond to the peaks observed on the fast protein liquid chromatography elution graph. To prepare negative stain grids, wrap a glass slide with filter paper with the carbon side facing up and place the grids that are going to be used for the sample preparation onto the slide.
Place the glass slide with the electron microscope grids into the chamber of a glow discharger between the two electrodes and replace the glass lid making sure the lid is centered and well-sealed. Run the glow discharging machine and make sure that the purple light generated by the plasma is visible. When the machine is finished running, wait until the chamber has reached atmospheric pressure before removing the glass lid and placing the slide onto the bench.
Adjust the concentration of the purified protein samples to about 0.1 milligrams per milliliter and load 3.5 microliters of the protein sample onto the 10 nanometer thick carbon grid. After one minute, use a piece of filter paper to remove the liquid from the electron microscopy grid surface and wash the grid surface three times with one three microliter droplet of water per wash. After the last water wash, wash the grid surface two more times with three microliter droplets of fresh filtered 2%uranium acetate per wash.
After the last uranium acetate wash, stain the grid with a three microliter droplet of fresh filtered 2%uranium acetate for one minute. At the end of the incubation, use a new piece of filter paper to remove the droplet and air dry the grid for at least one minute before storing the grid in a grid box for later use. Here, negative stain images obtained using transmission electron microscopy as demonstrated can be observed.
The negative stain image for the wild-type Acraflavin resistance channel protein B sample purified with detergent reveals a homogeneous solution of monodispersed particles with the protein displaying a well-defined trimeric quaternary structure. These trimeric structures correspond with the observed size exclusion chromatogram after purification. In comparison, in the negative stain image for the P223G mutant also purified with detergent, a heterogeneous solution of polydispersed nanoparticles with a propensity toward aggregation but no observable native trimers can be observed.
These results are also supported by size exclusion chromatography. Similar results were observed for wild-type and mutant proteins after purification with the membrane active polymer NCMNP11. The use of the NCMNP52 polymer facilitates the generation of native cell membrane nanoparticles in much larger sizes allowing multiple Acraflavin resistance channel protein B trimers to be imaged in a single native cell membrane particle.
In the mutant sample, however, no trimer particles are observed, even when looking at the large native cell membrane bilayer patches. SDS-PAGE analysis of the purified protein samples confirms the presence of Acraflavin resistance B in all of the samples with a greater than 95%purity at the predicted molecular weight of the protein. When modifying this protocol for other proteins, it is important to experimentally determine the appropriate amount of membrane fraction and the length and time and temperature for the solubilization process.
If the images of your sample reveal a homogenous solution of monodispersed particles with well-defined structural units, the sample can be used for high-resolution structure determination with cryo-electron microscopy.
