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Photoreaction pathways and photointermediates of retinal-binding photoreceptor proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy.

ABSTRACT: Photoirradiation solid-state NMR spectroscopy is a powerful means to study photoreceptor retinal-binding proteins by the detection of short-lived photointermediates to elucidate the photoreaction cycle and photoactivated structural changes. An in situ photoirradiation solid-state NMR apparatus has been developed for the irradiation of samples with extremely high efficiency to enable observation of photointermediates which are stationary trapped states. Such observation enables elucidation of the photoreaction processes of photoreceptor membrane proteins. Therefore, in situ photoirradiation is particularly useful study the photocycle of retinal-binding proteins such as sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) because functional photointermediates have relatively longer half-lives than other photointermediates. As a result, several photointermediates have been trapped as stationary state and their detailed structures and photoreaction cycles have been revealed using photoirradiation solid-state NMR spectroscopy at low temperature. Photoreaction intermediates of bacteriorhodopsin, which functions to provide light-driven proton pump activity, were difficult to trap because the half-lives of the photointermediates were shorter than those of sensory rhodopsin. Therefore, these photointermediates are trapped in a freeze-trapped state at a very low temperature and the NMR signals were observed using a combination of photoirradiation and dynamic nuclear polarization (DNP) experiments.

SUBMITTER: Naito A

PROVIDER: S-EPMC6441423 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Photoreaction pathways and photointermediates of retinal-binding photoreceptor proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy.

Naito Akira A Makino Yoshiteru Y Shigeta Arisu A Kawamura Izuru I

Biophysical reviews 20190227 2

Photoirradiation solid-state NMR spectroscopy is a powerful means to study photoreceptor retinal-binding proteins by the detection of short-lived photointermediates to elucidate the photoreaction cycle and photoactivated structural changes. An in situ photoirradiation solid-state NMR apparatus has been developed for the irradiation of samples with extremely high efficiency to enable observation of photointermediates which are stationary trapped states. Such observation enables elucidation of the ...[more]

PMID: 30811009

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Project description:Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N'-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N'-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at -60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N'- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N'-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N'-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N'-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.

Project description:Rhodopsin is a canonical member of class A of the G protein-coupled receptors (GPCRs) that are implicated in many of the drug interventions in humans and are of great pharmaceutical interest. The molecular mechanism of rhodopsin activation remains unknown as atomistic structural information for the active metarhodopsin II state is currently lacking. Solid-state (2)H NMR constitutes a powerful approach to study atomic-level dynamics of membrane proteins. In the present application, we describe how information is obtained about interactions of the retinal cofactor with rhodopsin that change with light activation of the photoreceptor. The retinal methyl groups play an important role in rhodopsin function by directing conformational changes upon transition into the active state. Site-specific (2)H labels have been introduced into the methyl groups of retinal and solid-state (2)H NMR methods applied to obtain order parameters and correlation times that quantify the mobility of the cofactor in the inactive dark state, as well as the cryotrapped metarhodopsin I and metarhodopsin II states. Analysis of the angular-dependent (2)H NMR line shapes for selectively deuterated methyl groups of rhodopsin in aligned membranes enables determination of the average ligand conformation within the binding pocket. The relaxation data suggest that the beta-ionone ring is not expelled from its hydrophobic pocket in the transition from the pre-activated metarhodopsin I to the active metarhodopsin II state. Rather, the major structural changes of the retinal cofactor occur already at the metarhodopsin I state in the activation process. The metarhodopsin I to metarhodopsin II transition involves mainly conformational changes of the protein within the membrane lipid bilayer rather than the ligand. The dynamics of the retinylidene methyl groups upon isomerization are explained by an activation mechanism involving cooperative rearrangements of extracellular loop E2 together with transmembrane helices H5 and H6. These activating movements are triggered by steric clashes of the isomerized all-trans retinal with the beta4 strand of the E2 loop and the side chains of Glu(122) and Trp(265) within the binding pocket. The solid-state (2)H NMR data are discussed with regard to the pathway of the energy flow in the receptor activation mechanism.

Dataset Information

Photoreaction pathways and photointermediates of retinal-binding photoreceptor proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy.

Publications

Photoreaction pathways and photointermediates of retinal-binding photoreceptor proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy.

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