Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy.
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 process...
| Publicado en: | Journal of the American Chemical Society Vol. 138; no. 43; pp. 14238 - 14242 |
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| Autores principales: | , , , , |
| Formato: | Artículo |
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American Chemical Society
11/2/2016
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=119789619&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 119789619 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 11/2/2016 vid: 138 iid: 43 pid: 997 pub: American Chemical Society artinfo: ui: 119789619 10.1021/jacs.6b09542 ppf: 14238 ppct: 4 formats: tig: atl: Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy. aug: au: Janghyun Yoo Tae-Sun Lee Byungsan Choi Min Ju Shon Tae-Young Yoon affil: 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 su: Protein-protein interactions Single molecules Fluorescence microscopy Dissociation (Chemistry) Molecular biology Optical diffraction sug: subj: Protein-protein interactions Single molecules Fluorescence microscopy Dissociation (Chemistry) Molecular biology Optical diffraction ab: 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 singlemolecule 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. pubtype: Academic Journal doctype: Article src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2016 holdings: @attributes: islocal: N |
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