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Chimeric microbial rhodopsins for optical activation of Gs-proteins.

ABSTRACT: We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of ?2-adrenergic receptor (?2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins.

SUBMITTER: Yoshida K

PROVIDER: S-EPMC5774426 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Chimeric microbial rhodopsins for optical activation of Gs-proteins.

Yoshida Kazuho K Yamashita Takahiro T Sasaki Kengo K Inoue Keiichi K Shichida Yoshinori Y Kandori Hideki H

Biophysics and physicobiology 20171219

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and <i>Gloeobacter</i> rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microb ...[more]

PMID: 29362703

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Project description:G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. Microbial rhodopsins possess an all-trans retinal, and all-trans to 13-cis photoisomerization triggers protein structural changes for each function. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. However, it was reported that bacteriorhodopsin (BR) chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein, suggesting a common mechanism of protein structural changes. Here we design chimeric proteins for Natronomonas pharaonis sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin), which has a two-orders-of-magnitude slower photocycle than BR. Light-dependent transducin activation was observed for most of the nine SRII chimeras containing the third cytoplasmic loop of bovine Rh (from Y223, G224, Q225 to T251, R252, and M253), but the activation level was 30,000-140,000 times lower than that of bovine Rh. The BR chimera, BR/Rh223-253, activates a G-protein transducin, whereas the activation level was 37,000 times lower than that of bovine Rh. We interpret the low activation by the chimeric proteins as reasonable, because bovine Rh must have been optimized for activating a G-protein transducin during its evolution. On the other hand, similar activation level of the SRII and BR chimeras suggests that the lifetime of the M intermediates is not the simple determinant of activation, because SRII chimeras have two-orders-of-magnitude's slower photocycle than the BR chimera. Activation mechanism of visual and microbial rhodopsins is discussed on the basis of these results.

Dataset Information

Chimeric microbial rhodopsins for optical activation of Gs-proteins.

Publications

Chimeric microbial rhodopsins for optical activation of Gs-proteins.

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