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.

Interactions between signal-transducing proteins measured by atomic force microscopy.

Atomic force microscopy (AFM) has been used to study the specific interactions between the signal-transducing proteins mammalian phospholipase D1 (PLD1), phospholipase C-gamma1 (PLC-gamma1), and Munc-18-1. To record the forces between them, the Phox homology (PX) domain of PLD1, the Src homology (SH3) domain of PLC-gamma1, and Munc-18-1 were fused with glutathione S-transferase (GST) and immobilized onto reduced glutathione (GSH)-tethered surfaces. In order to enhance the recognition efficiency and avoid undesirable complications, both AFM tips and substrates were first modified with dendrons of two different sizes.