Comparing the Affinity of GTPase-binding Proteins using Competition Assays

The overall goal of this procedure is to compare the relative affinities of protein binding partners for Row Family GT P ACEs under carefully controlled nucleotide loading conditions. This is accomplished by first purifying putative competitive binding partners, each labeled with the same tag, for example, green fluorescent protein or GFP. The second step is to purify the GTPA of interest.

For example, RAC one and load with the appropriate nucleotide to achieve the desired GTPA signaling state. Next, the nucleotide loaded gtpa is incubated with a fixed amount of one binding competitor and increasing amounts of the other binding competitor to achieve a titration binding curve. The final step is to resolve proteins bound to immobilized GT pase by western blot and probe the membrane for the GFP tag that is shared between the two binding partners.

Ultimately, the Western blot is used to identify the relative concentrations at which similar amounts of each GFP tagged binding partner is captured by the GTPase. The main advantage of this technique over existing methods like co precipitation, is that the protein binding properties of gtpa is a dictated by the bound nucleotide in the cell. The bound nucleotide is labile making interpretation of protein binding data difficult.

Begin this procedure with purification of GST tagged G tpa and expression of gtpa binding proteins as detailed in the text protocol. RINs the flasks of transfected cells in phosphate buffer saline or PBS and drain the flask for five minutes. Aspirating the free liquid, then scrape off the cells in 500 microliters of lysis buffer into a micro fuge tube, mixed cells to lyse by inversion at four degrees Celsius for 30 minutes.

During the lysis wash two lots of 40 microliters of GFP trap beads three times with fresh lysis buffer sediment. The beads at 2, 700 times G for two minutes between washes. Following lysis, clarify the lysates by centrifugation at 21, 000 times G for 10 minutes.

Transfer the clarified lysate of each of the competitor proteins to separate washed GFP trap beads. Allow GFP fusion proteins to bind for two hours, mixing by inversion at four degrees Celsius. Wash the loaded GFP trap beads twice in lysis buffer and twice in competition.

Binding buffer sedimenting beads at 2, 700 times G for two minutes between washes. Elute GFP fusion proteins by adding 40 microliters of 0.2 molar glycine and pipetting up and down for 30 seconds immediately sediment the beads at 21, 000 times G for 60 seconds and transfer the liquid to a new fuge tube containing four microliters of one molar tris HCL perform this step quickly to limit damage to the purified protein. Analyze one microliter of each purified protein by western blot and probe with an anti GFP antibody to establish the relative yield using a quantitative blotting system according to the manufacturer's protocol.

Equalize molar protein concentration by addition of competition binding buffer. Take 90 microliters of prepared GST rack one beads and wash three times with nucleotide loading buffer using a magnetic particle sorter to precipitate the beads at each step. Aspirate the buffer from the beads and add 100 microliters of nucleotide loading buffer according to whether G-D-P-G-T-P or no nucleotide loading is required.

For the competition experiment, add 12 microliters of 100 millimolar GDP 12 microliters of 10 millimolar GTP gamma S or no nucleotide to 60 microliters of GST rack one beads for the nucleotide loading controls. Split the remaining beads into three 10 microliter quats and add two microliters of 100 millimolar GDP two microliters of 10 millimolar GTP gamma S or no nucleotide to each tube. Incubate the bead mixes for 30 minutes at 30 degrees Celsius with agitation.

Stabilize the nucleotide bound rack, one by addition of one molar magnesium chloride to the experimental mix and the control mixes To perform the competition binding. Set up six micro fuge tubes. Each containing 200 microliters of competition binding buffer.

Each tube should also contain 10 microliters of the nucleotide loaded rack, one beads and five microliters of the rack. One binding protein A as a constant binding protein to each tube add 0 1 2 0.55, 10, or 20 microliters of rack one binding protein B as the variable binding protein. These volumes assume approximately equal stock concentrations of the constant and variable binding proteins and may need to be adjusted.

Make up the total volume of the binding mixture to 235 microliters by addition of the competition binding buffer. Next, set up a micro fuge tube containing 200 microliters of the competition buffer. 10 microliters of experimental nucleotide loaded rack, one beads and 10 microliters of rack one binding protein A.Then set up the G-D-P-G-T-P gamma S and no nucleotide control tubes as described in the text protocol.

Incubate the tubes for two hours mixing by inversion at four degrees Celsius following incubation. Wash the beads three times with the competition binding buffer. Finally, elute the bound proteins in 20 microliters of reducing sample buffer.

Quantitative western blotting of the GFP tag of purified GFP RCC two and GFP Corona in one C.Propeller domain reveals that the protein in the upper band, GFP RCC two is 1.4 times more abundant than the lower band and should be diluted to achieve a one-to-one molar ratio for the competition experiment. A control experiment demonstrates that the nucleotide loading status of rack one dictates the binding specificity titrating increasing volumes of GFP RCC two against a fixed volume of equimolar GFP Corona in one C propeller domain and blotting. The proteins that bind to rack one allows a relative change in bound protein to be visualized plotting the GFP band intensities for both competitors on the same graph, and identifying the point at which the lines intersect, allows the volume of variable binding protein at equilibrium to be identified.

However, care must be taken to ensure that issues such as interaction between competitors or excess bait gtpa do not compromise the experiment. Increase in one competitor without loss of the other as shown here, indicates a problem While attempting this procedure. It's important to remember to check that there is a reciprocal relationship between the abundance of the competing binding partners.

There are a number of issues that can distort the results and or can be resolved as long as they're identified by inspecting the data.