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).

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. Under the employed conditions, the probability of observing an unbinding event increased, most force-distance curves showed the single rupture events, and the unbinding forces were 51 +/- 2 pN for PX-(Munc-18-1) and 42 +/- 2 pN for PX-SH3. To investigate dynamics of these biomolecular interactions, we measured the loading rate dependence of the unbinding forces. The unbinding forces increased linearly with the logarithm of the loading rate, indicating the presence of a single potential barrier in the dissociation energy landscape. The measured off-rate constants (k(off)) at 15 degrees C were 10(-3.4 +/- 0.3) s(-1) for PX-(Munc-18-1) and 10(-1.7 +/- 0.1) s(-1) for PX-SH3. Further, we elucidated the influence of free SH3 and Munc-18-1 on the specific PX-(Munc-18-1) and PX-SH3 interaction, respectively.