title: Interactions between signal-transducing proteins measured by atomic force microscopy.
abstract: 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.
authors.affiliation: National Core Research Center for Systems Bio-Dynamics, Department of Chemistry, Pohang University of Science and Technology, San 31 Hyoja-dong, Pohang, 790-784, Korea.
authors.forename: Il Hong
authors.initials: IH
authors.lastname: Kim
authors.forename: Hye Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Hae Dong
authors.initials: HD
authors.lastname: Lee
authors.forename: Yu Jin
authors.initials: YJ
authors.lastname: Jung
authors.forename: Saul J B
authors.initials: SJ
authors.lastname: Tendler
authors.forename: Philip M
authors.initials: PM
authors.lastname: Williams
authors.forename: Stephanie
authors.initials: S
authors.lastname: Allen
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Ryu
authors.forename: Joon Won
authors.initials: JW
authors.lastname: Park