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Biophys J. 2014 Feb 18;106(4):915-24. doi: 10.1016/j.bpj.2014.01.020.

Single-molecule observation of the ligand-induced population shift of rhodopsin, a G-protein-coupled receptor.

Author information

1
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
2
Cellular Informatics Laboratory, RIKEN, Wako, Japan; Laboratory for Cell Signaling Dynamics, RIKEN Quantitative Biology Center, Furuedai, Suita, Japan.
3
Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe, Japan.
4
Cellular and Structural Physiology Institute, Nagoya University, Nagoya, Japan.
5
Cellular Informatics Laboratory, RIKEN, Wako, Japan.
6
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan. Electronic address: imamoto@rh.biophys.kyoto-u.ac.jp.

Abstract

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.

PMID:
24559994
PMCID:
PMC3945020
DOI:
10.1016/j.bpj.2014.01.020
[Indexed for MEDLINE]
Free PMC Article

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