Nanoscale Mapping and Affinity Constant Measurement of Signal-transducing Proteins by Atomic Force Microscopy

Atomic force microscope (AFM) was used to measure the interaction force between two signal-transducing proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Ras homologue enriched in brain (Rheb), and to analyze the binding of glyceraldehyde-3-phosphate (Gly-3-P) to GAPDH. To enhance the recognition efficiency and avoid undesirable multiple interactions, the AFM probe and the substrate were each modified with a dendron, glutathione S-transferase (GST)-fused proteins were employed, and reduced glutathione (GSH) was conjugated at the apex of each immobilized dendron. The resulting median specific force between GAPDH and Rheb was 38 ± 1 pN at a loading rate of 3.7 × 10(3) pN/s. The measurements showed that the GAPDH-Rheb interaction was inhibited by binding of Gly-3-P. An adhesion force map showed individual GADPHs on the surface and that the number density of GAPDH decreased with the concentration of Gly-3-P. Maps obtained in the presence of various Gly-3-P concentrations provided information on the binding behavior, yielding a thermodynamic association constant of 2.7 × 10(5) M(-1).