This article discusses the competitive binding of proteins to GTPases, specifically focusing on the interaction between fluorescently-tagged proteins and GTP-bound GTPases. The methodology involves using magnetic beads to study the binding affinities of different proteins.
Guanine nucleotide-binding proteins — a category of GTPases — function as a molecular switch, cycling between a GTP-bound active form and a GDP-bound inactive form.
The active GTPases bind to specific protein partners. These partners compete for the same binding site on the GTPases.
To study competitive binding, begin by taking a suspension of GTP-bound GTPase immobilized on magnetic beads. Add the first interaction partner — fluorescently-tagged protein A. Incubate under agitation to keep the beads in suspension. Protein A binds and saturates the binding sites on all the GTPase molecules.
Partition this solution equally into multiple tubes. Add a second interaction partner — fluorescently-tagged protein B — to the tubes in increasing amounts. Incubate under agitation.
Protein B binds to the same binding site on the GTPases — competitively displacing protein A.
Expose the tubes to a magnetic field to collect the beads on one side. Discard the supernatant to remove unbound proteins. Resuspend the protein-complexed beads in a suitable buffer, and quantify the bound proteins.
Plot the relative intensities of proteins A and B bound to GTPase at each concentration of protein B.
Protein B reaches an equilibrium — occupying the binding sites of half of the GTPase molecules — at a relatively lower concentration than protein A, indicating the higher affinity of protein B for GTPase.
To perform the competition binding, set up six microfuge tubes each containing 200 microliters of competition binding buffer. Each tube should also contain 10 microliters of the nucleotide-loaded Rac1 beads and five microliters of the Rac1-binding protein A as a constant binding protein.
To each tube, add 0, 1, 2.5, 5, 10, or 20 microliters of Rac1-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 microfuge tube containing 200 microliters of the competition buffer, 10 microliters of experimental nucleotide-loaded Rac1 beads, and 10 microliters of Rac1-binding protein A.
Then, set up the GDP, GTPγS, and no nucleotide control tubes as described in the text protocol. Incubate the tubes for two hours, mixing by inversion at 4 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.
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