Interaction between Signal Transducing Proteins Measured by Picoforce Atomic Force Microscopy
I.H. Kim, H.Y. Lee, Y.J. Jung, S.H. Ryu and J.W. Park
Center for Integrated Molecular Systems, KR
dendron, protein, bio-AFM
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