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Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy.

ABSTRACT: Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state (2)H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. Moreover (2)H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state (2)H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition we review optimized conditions for trapping the rhodopsin photointermediates; and we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films.

SUBMITTER: Struts AV

PROVIDER: S-EPMC5270617 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy.

Struts Andrey V AV Chawla Udeep U Perera Suchithranga M D C SM Brown Michael F MF

Methods in molecular biology (Clifton, N.J.) 20150101

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state (2)H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. Moreover (2)H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the acti ...[more]

PMID: 25697522

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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

Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy.

Publications

Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy.

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