Janghyun Yoo†‡§, Tae-Sun Lee‡§, Byungsan Choi†‡§, Min Ju Shon‡§, and Tae-Young Yoon*‡§
† Department of Physics, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea
‡ Center for Nanomedicine, Institute for Basic Science (IBS), Yonsei University, Seoul 30722, South Korea
§ Yonsei-IBS Institute, Yonsei University, Seoul 30722, South Korea
Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant. Instead of increasing the concentration of diffusing molecules, which is inevitably limited by the optical diffraction limit, we employed an increased density of surface bait protein. The low occupancy of the surface baits permitted determination of the kinetics with single-molecule resolution. We used this approach to study a PPI network consisting of Ras and its downstream proteins including full-length Rafs and catalytic subunits of phosphoinositide 3-kinase.