Interaction between Signal Transducing Proteins Measured by Picoforce Atomic Force Microscopy

Keywords:
dendron, protein, bio-AFM

Abstract:
Picoforce atomic force microscopy (AFM) has been used to study specific interaction between signal transducing proteins, mammalian phospholipase D1 (or PLD1), phospholipase C- 1 (or PLC- 1), and Munc-18-1.1,2 To record the force between them, Phox homology (PX) domain of PLD1, Src homology (SH) 3 domain of PLC- 1, and Munc-18-1 were specifically fused with GST, and the GST-fused proteins were immobilized onto the GSH tethered surface. In order to enhance the recognition efficiency and avoid undesirable multiple interactions, both AFM tip and substrate were modified a dendron with relevant size (Figure 1).3 Under the condition, increased unbinding event probability and force-distance curves showing single rupture were observed. We elucidated the influence of SH3 and Munc-18-1 in the specific PX-(Munc-18-1) interaction and PX-SH3 interaction, respectively. The unbinding force of the protein pairs decreased as the concentration of SH3 and Munc-18-1 increases, and the concentration of SH3 and Munc-18-1 required to reduce the pristine value into half, c1/2, is 38 nM and 6.8 nM, respectively. Also, temporal behavior of the force upon addition of SH3 or Munc-18-1 suggests that the reorganization process of PX domain induced by SH3 is relatively slow while the process by Munc-18-1 is relatively fast.

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Nanotech 2007 Conference Program Abstract