title: Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics.
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.
authors: National Creative Research Initiative Center for Single-Molecule Systems Biology, KAIST, Daejeon 305-701, South Korea.
authors: Hong-Won
authors: HW
authors: Lee
authors: Taeyoon
authors: T
authors: Kyung
authors: Janghyun
authors: J
authors: Yoo
authors: Tackhoon
authors: T
authors: Kim
authors: Chaeuk
authors: C
authors: Chung
authors: Ji Young
authors: JY
authors: Ryu
authors: Hanki
authors: H
authors: Lee
authors: Kihyun
authors: K
authors: Park
authors: Sangkyu
authors: S
authors: Lee
authors: Walton D
authors: WD
authors: Jones
authors: Dae-Sik
authors: DS
authors: Lim
authors: Changbong
authors: C
authors: Hyeon
authors: Won Do
authors: WD
authors: Heo
authors: Tae-Young
authors: TY
authors: Yoon
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003199:Computer Systems; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D015398:Signal Transduction; D023041:Xenograft Model Antitumor Assays; D018631:ras Proteins / Q000378:metabolism*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins