Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

The overall goal of this procedure is to visualize the binding of an extrinsic membrane protein on a nanodisc membrane surface by negative stain transmission electron microscopy as a first step towards high resolution structure determination of the protein. This method can help answer key questions in several research fields as it concerns protein activities that occur in and on cellular membranes. The main advantage of this technique is the characteristic stacks of nanodisc forms if the protein cannot bind to the membranes.

These stacks are clearly visible by transmission electron microscopy. The implication of this technique extends towards drug development as compounds can be easily screened for the ability to block or enable the membrane protein interactions. Though this method can provide insight into the optimal conditions for monotopic protein binding to your membrane, it also provides a low resolution structure of the protein nanodisc complex.

To begin the procedure, express and purify membrane scaffolding protein such as MSP1E3D1. Next, using a glass syringe with a metal needle, dispense 305 microliters of 25 milligrams per milliliter of POPC in chloroform into a glass round-bottom flask. Evaporate the chloroform under a gentle stream of nitrogen gas in a fume hood.

Dry the remaining lipid overnight in a vacuum desiccator. Then dissolve the dried lipid cake in 200 microliters of MSP standard buffer enriched with 100 millimolar sodium cholate as detergent. Vortex the mixture until transparent to obtain a suspension of 50 millimolar POPC in buffer.

Next, wash five grams of hydrophobic beads with 30 milliliters of 100%methanol then with 40 milliliters of ultrapure water and finally with 10 milliliters of MSP standard buffer. Store the beads under 15 milliliters of standard buffer at four degrees Celsius. Then combine 190 microliters of a 0.124 millimolar solution of MSP1E3D1 and 61.5 microliters of a 50 millimolar suspension of POPC in buffer resulting in a one to 130 molar ratio of MSP to POPC and a sodium cholate concentration of 25 millimolar.

The ideal ratio of the lipid per MSP varies with each choice of lipid type and MSP lens and must be optimized to obtain a homogenous preparation of nanodiscs. Incubate the mixture on wet ice for one hour. Then add the solution to a tube containing 0.5 grams of washed hydrophobic beans per milliliter of reconstitution mixture to initiate nanodisc self assembly.

Incubate the mixture at four degrees Celsius for 16 hours at seven to eight rotations per minute. After incubation, allow the beads to settle by gravity. Remove and store the supernatant at four degrees Celsius until ready to perform size-exclusion chromatography.

Prior to size-exclusion chromatography, centrifuge the mixture at 13, 000 g for 10 minutes at four degrees Celsius. Decant the supernatant and discard the pellet. Equilibrate a size-exclusion chromatography column with MSP standard buffer until the baseline at 280 nanometers is stable.

Inject the supernatant onto the column and collect the product in 0.5 milliliter fractions. Measure the absorbance of the fractions at 280 nanometers with a UV vis spectrophotometer. Calculate the concentration of nanodiscs using the molar extinction coefficient for the chosen MSP.

To perform non-denaturing gel electrophoresis, first mix 15 microliters of the sample with five microliters of the appropriate loading buffer. Fill the cathode tank with light cathode buffer and the anode tank with running buffer. Load the sample onto a four to 16%Bis-Tris gel and begin the run.

Stain the gel per the gel manufacturer's instructions. To begin preparation of a nanodisc monotopic protein complex with five lipoxygenase as the protein, prepare a batch of MSP standard buffer enriched with 1.5 millimolar calcium chloride. Express and purify the 5LO protein.

Immediately prepare a 100 microliter mixture of 0.8 micromolar 5LO and 0.8 micromolar nanodiscs in calcium-enriched MSP standard buffer. Our example of a monotopic proteinase five lipoxygenase depends on calcium amounts to bind to the membrane. 5LO is highly sensitive and must be used within a few hours of preparation.

Incubate the mixture on ice for 10 minutes. Store the resulting nanodisc protein complex sample at four degrees Celsius for up to one month. To begin preparation for the TEM analysis, dissolve one gram of phosphotungstate sodium salt in 50 milliliters of ultrapure water at room temperature.

Adjust the solution pH to 7.4 with a one molar solution of sodium hydroxide. Filter the phosphotungstate sodium solution through a 0.22 micrometer syringe filter and store the solution at room temperature. Next, glow discharge of 400 mesh carbon coated copper grid for 20 seconds at 30 milliamps to render the grid surface hydrophilic.

Place between 2.5 and five microliters of the nanodisc monotopic protein complex sample on the grid and allow the sample to sit for 30 seconds. Then use filter paper to blot excess solution from the grid. Immediately apply a drop of phosphotungstate sodium solution and let the solution sit for 30 seconds.

Blot the excess solution and allow the grid to air dry. Perform transmission electron microscopy with an accelerated voltage of 120 to 200 kilovolts. Exclude images showing long stacks from subsequent image processing.

For the selected images, use standard processing methods to determine the class averages and to generate a low resolution 3D model of the nanodisc monotopic protein. Using this method, empty nanodiscs were prepared with a one to 130 ratio of membrane scaffolding proteins to lipids. Only one major peak was observed during size-exclusion chromatography and only one band was observed in blue native gel electrophoresis.

When the empty nanodiscs are treated with a phosphotungstate sodium salt solution, stacking is induced. These long stacks can then be observed by TEM. This stacking was not disrupted by the inclusion of calcium ions in the nanodisc solution before application of the phosphotungstate sodium solution.

When a monotopic protein such as five lipoxygenase is bound to the nanodisc surface, stacking is hindered by stearic obstruction by the protein. Both sides of the nanodisc are available for binding allowing formation of one to one and two to one 5LO nanodisc complexes. The sample of two to one 5LO nanodisc complexes exhibited less stacking than the one to one sample.

5LO binding requires the presence of calcium ions. This was confirmed by observation of stacking in a mixture of 5LO and nanodiscs without calcium which indicated that 5LO had not bound to the membrane surfaces. Once the monotopic protein and the nanodiscs are prepared, evaluation of protein binding to your membrane can be done in an afternoon if it is performed properly.

While attempting this procedure, it is important to estimate the area of the protein on the nanodisc to choose the correct MSP length. For well-matched sizes, maximally two extrinsic proteins may bind one on each side of the disc. It is also important that the sodium salt of phosophotungsten is used for the negative stand to ensure maximal stacking as binding is confirmed by comparison of the absence of stacks to the long stacks of a sample without a monotopic protein.

Use of the nanodiscs instead of liposomes enables the use of dynamic light scattering, small angle x-ray scattering to answer additional questions about sample homogeneity size or molecular structure. The low resolution 3D structure of a monotopic membrane protein bound to a nanodisc could be an easily accessible first step in the path to high resolution structure of those bound proteins.