Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

Hong-Won Lee, Taeyoon Kyung, Janghyun Yoo, Tackhoon Kim, Chaeuk Chung, Ji Young Ryu, Hanki Lee, Kihyun Park, Sangkyu Lee, Walton D. Jones, Dae-Sik Lim, Changbong Hyeon, Won Do Heo () and Tae-Young Yoon ()
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Hong-Won Lee: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST
Taeyoon Kyung: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST
Janghyun Yoo: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST
Tackhoon Kim: KAIST
Chaeuk Chung: KAIST
Ji Young Ryu: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST
Hanki Lee: BioNanotechnology Research Center, KRIBB
Kihyun Park: School of Computational Sciences, Korea Institute for Advanced Study
Sangkyu Lee: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST
Walton D. Jones: KAIST
Dae-Sik Lim: KAIST
Changbong Hyeon: School of Computational Sciences, Korea Institute for Advanced Study
Won Do Heo: KAIST
Tae-Young Yoon: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST

Nature Communications, 2013, vol. 4, issue 1, 1-9

Abstract: Abstract Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level.

Date: 2013
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DOI: 10.1038/ncomms2507

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