Retinal conformation and dynamics in activation of rhodopsin illuminated by solid-state H NMR spectroscopy

Abstract

Solid-state NMR spectroscopy gives a powerful avenue for investigating G protein-coupled receptors and other integral membrane proteins in a native-like environment. This article reviews the use of solid-state (2)H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site-specific (2)H NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability (2)H NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three-fold (C(3)) axes with an order parameter for the off-axial motion of SC(3) approximately 0.9. For the dark state, the (2)H NMR structure of 11-cis-retinal manifests torsional twisting of both the polyene chain and the beta-ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11=C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11-cis to trans isomerization. In addition, (2)H NMR has been applied to study the retinylidene dynamics in the dark and light-activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the beta-ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the beta-ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid-state (2)H NMR thus provides information about the flow of energy that triggers changes in hydrogen-bonding networks and helix movements in the activation mechanism of the photoreceptor.

Figure 1

Powder-type ²H NMR spectra for rhodopsin with ²Hlabeled retinal in the dark state indicate rotating methyl groups with large order parameters. (a–c) Experimental ²H NMR spectra for 11-Z-[9-C²H₃]-retinylidene rhodopsin, i.e. having 11-cis-retinal deuterated at the C9-methyl group, in gel-phase POPC membranes (1:50 molar ratio). (d) Theoretical ²H NMR spectrum for randomly oriented C–C²H₃ groups undergoing rapid three-fold rotation on the ²H NMR time scale (<(3_χQ/8)⁻¹ ≈ 10 μs). (e, f) Representative ²H NMR spectra for dark-state 11-Z-[5-C²H₃]-retinylidene rhodopsin and 11-Z-[13-C²H₃]-retinylidene rhodopsin, i.e. with 11-cis-retinal deuterated at the C5-methyl (yellow) or C13-methyl group (red), respectively, in POPC membranes (1:50). Theoretical ²H NMR spectra for C–C²H₃ groups undergoing axial rotation (continuous color lines) are superimposed on the experimental spectra, with residuals below. Adapted with permission from Struts et al. (64).

Figure 2

Orientation-dependent ²H NMR spectra for aligned rhodopsin POPC (1:50) recombinant membranes provide angular restraints for retinylidene ligand in the dark state. (a–c) ²H NMR spectra for 11-Z-[5-C²H₃]-retinylidene rhodopsin (blue), 11-Z-[9-C²H₃]-retinylidene rhodopsin (magenta) and 11-Z-[13-C²H₃]-retinylidene rhodopsin (green) at pH 7 and T = −150°C. Theoretical lineshapes for an immobile uniaxial distribution (solid lines) are superimposed on the experimental ²H NMR spectra. Note that characteristic lineshape changes are observed as a function of the tilt angle, which manifest the different methyl bond orientations with respect to the membrane frame. Reproduced with permission from Struts et al. (64).

Figure 3

Global fitting of ²H NMR spectra for 11-cis-retinal in the dark state of rhodopsin gives methyl bond orientations and mosaic spread of aligned membranes. (a–c) RMSD of calculated versus experimental ²H NMR spectra for retinal deuterated at C5-, C9- or C13-methyl groups, respectively and (d–f) cross-sections through hypersurfaces. Distinct minima are found in the bond orientation θ_B and mosaic spread σ of aligned membranes. Reproduced with permission from Struts et al. (64).

Figure 4

Solid-state ²H NMR spectroscopy yields conformation and orientation of 11-cis-retinal ligand in the dark state of rhodopsin. Retinal is described by three planes of unsaturation (designated A, B, C). (a) Simple three-plane model with torsional twisting only about C6–C7 and C12–C13 bonds. (b) Extended three-plane model with additional pretwisting about the C11=C12 double bond. Reproduced with permission from Struts et al. (64).

Figure 5

Solid-state NMR provides the structure of retinal ligand of the canonical GPCR rhodopsin. Vertical direction corresponds to the membrane normal, where the extracellular side is at top and the cytoplasmic side is below. The ²H NMR structure of 11-cis-retinal in the dark state (green) is compared to retinal structure in the preactivated metarhodopsin I state (red). Figure prepared with PyMOL [http://pymol.sourceforge.net/].

Figure 6

Structure and dynamics of retinal within the binding cavity of rhodopsin in the dark state at −60°C. Analysis of ²H NMR data reveals torsional twisting of retinal that accompanies nonbonded interactions of the C5- and C13-methyls with the polyene B-plane (for retinal numbering, see Fig. 1). Average structure of the retinylidene ligand corresponds to a distorted 6-s-cis,11-cis,12-s-trans,15-anti conformation. Rapid spinning of the methyl groups about their three-fold axes occurs with correlation times in the range ≈ 1–20 ps and off-axial order parameters of S_C3 ≈ 0.9. The rates of three-fold rotation (in GHz) of the C5-, C9- and C13-methyl groups and the corresponding activation energies (in kJ mol⁻¹) are indicated in the figure. Methyl rotation may be coupled to fluctuations of the C6–C7 and C12–C13 dihedral angles which connect the different planes of unsaturation (A, B and C). Correlation times and activation barriers for fluctuations of the C5-methyl of the β-ionone ring are considerably greater than for the polyene C9- or C13-methyl groups. The C9-methyl group may be implicated in the activation process through a nearly frictionless environment manifested by the low activation barrier. We propose that site-specific differences in mobility of the retinal ligand underlie movement of retinal in the activation mechanism of rhodopsin.