Single-molecule co-IP for quantitative protein-protein interaction analysis

Abstract

Weak and transient protein-protein interactions, that can show fast response to the cell environment change, are a key to understand cell signaling. However, their weakly interacting nature followed by high substrate concentration of sub-μM to hundreds of μM requirement in detection makes them hard study. Single-molecule imaging has high detection sensitivity. But diffraction limited observation volume makes its concentration barrier at nM range. Few approaches have successfully broken through the concentration barrier. However, in this study, instead of breaking the concentration barrier, we will bypass it through twisting the imaging scheme without major modification in conventional single-molecule imaging system. Increased bait density allowed us to monitor interactions at low prey concentration which is four orders of magnitude lower than its dissociation constant. It enabled us to quantify intertwined kinetics of signaling proteins: Ras, Raf and Raf dimers. This new scheme can be extended to study kinetics that requires tens of mM range or even further.