Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

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.