26 research outputs found
Single-molecule observation of the ligand-induced population shift of rhodopsin, a g-protein-coupled receptor.
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
Shift in Conformational Equilibrium Induces Constitutive Activity of G-Protein-Coupled Receptor, Rhodopsin
Constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) cause various kinds of diseases. Rhodopsin, a light-absorbing GPCR in animal retinas, has retinal as an endogenous ligand; only very low levels of activation of G-protein can be obtained with the ligand-free opsin. However, the CAM of opsin activates G-protein much more efficiently than the wild type, but the mechanism underlying this remains unclear. The present work revisits the constitutive activity of rhodopsin from the standpoint of conformational dynamics. Single-molecule observation of the M257Y mutant of bovine rhodopsin demonstrated that the switch between active and inactive conformations frequently occurred in M257Y opsin, and frequent generation of the active state results in the population shift toward the active state, which accounts for the constitutive activity of M257Y opsin. Our findings demonstrate that the protein function has a direct connection with the structural dynamics
Shift in Conformational Equilibrium Induces Constitutive Activity of G‑Protein-Coupled Receptor, Rhodopsin
Constitutively active mutants (CAMs)
of G-protein-coupled receptors
(GPCRs) cause various kinds of diseases. Rhodopsin, a light-absorbing
GPCR in animal retinas, has retinal as an endogenous ligand; only
very low levels of activation of G-protein can be obtained with the
ligand-free opsin. However, the CAM of opsin activates G-protein much
more efficiently than the wild type, but the mechanism underlying
this remains unclear. The present work revisits the constitutive activity
of rhodopsin from the standpoint of conformational dynamics. Single-molecule
observation of the M257Y mutant of bovine rhodopsin demonstrated that
the switch between active and inactive conformations frequently occurred
in M257Y opsin, and frequent generation of the active state results
in the population shift toward the active state, which accounts for
the constitutive activity of M257Y opsin. Our findings demonstrate
that the protein function has a direct connection with the structural
dynamics