Abstract
Single-molecule kinetic and dynamic analyses of the biochemical reactions in living cells are one of the most useful methods of investigating the molecular mechanisms of cellular reactions, especially those involved in signal transduction on the plasma membrane. Here, we focus on single-molecule analyses of the intracellular signaling from RAS to RAF, which occurs on the plasma membrane. RAS and RAF are cell signaling proteins that regulate various cellular behaviors, including cell fate determination for proliferation and differentiation. Using single-molecule techniques in living cells, including single-pair Förster resonance energy transfer (FRET) detection and single-molecule tracking, we directly measured the elementary processes in the reactions between RAS and RAF, including the dissociation of RAS and RAF, the conformational changes in RAF, and the diffusion of RAS and RAF along the plasma membrane. Based on the results of these measurements, we discuss how the mutual molecular recognition between RAS and RAF facilitates accurate signal transduction.
Keywords
- Conformational change
- Cysteine-rich domain (CRD)
- Diffusion coefficient
- Dissociation
- Effector protein
- Epidermal growth factor (EGF)
- Fluorescence emission spectrum
- GDP–GTP binary molecular switch
- Green fluorescent protein (GFP)
- HeLa cell
- Induced conformational change
- Initial binding state
- Intracellular signaling
- Lateral diffusion
- Membrane ruffle
- Mutual molecular recognition
- Objective-type total internal reflection fluorescence (TIRF) microscopy
- Plasma membrane
- RAF
- RAS
- RAS-binding domain (RBD)
- Serine/threonine kinase
- Single-molecule kinetic and dynamic analyses
- Single-molecule tracking
- Single-pair Förster resonance energy transfer (FRET)
- Small GTPase
- Translocation
- Yellow fluorescent protein (YFP)
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Acknowledgments
We would like to thank our collaborators, Tatsuo Shibata (Hiroshima University) and Toshio Yanagida (Osaka University), and the members of our laboratory.
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Hibino, K., Sako, Y. (2011). Single-Molecule Analysis of Molecular Recognition Between Signaling Proteins RAS and RAF. In: Sako, Y., Ueda, M. (eds) Cell Signaling Reactions. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9864-1_3
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DOI: https://doi.org/10.1007/978-90-481-9864-1_3
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