Solid-state 2H NMR spectroscopy of retinal proteins in aligned membranes

Abstract

Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions.

Fig. 1

Powder-type ²H NMR spectra for rhodopsin with ²H-labeled 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 3-fold rotation on the ²H NMR time scale (< (3χ_Q/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 Ref. [77].

Fig. 2

Solid-state NMR yields structural information for ligands bound to integral membrane proteins. (a) Illustration of nuclear spin energy levels obtained by solving the Schrödinger equation for an ²H system [25]. Degeneracy of the single quantum transitions due to the Zeeman interaction (Ĥ_Z) is removed by the orientation-dependent quadrupolar coupling (Ĥ_Q). The frequency separation between the single-quantum quadrupolar frequencies is the (residual) quadrupolar coupling Δν_Q. (b) As an illustration we show the retinal chromophore of bacteriorhodopsin in membranes as investigated by solid-state ²H NMR spectroscopy. Geometry of the tilt experiments is presented for a static uniaxial distribution of aligned bilayers. For a given methyl group, θ_B is angle of C–C²H₃ bond axis to the local membrane normal n, with static rotational symmetry given by the azimuthal angle ϕ. Alignment disorder is described by angle θ′ of n relative to the average membrane normal n₀, and is likewise uniaxially distributed as characterized by ϕ′. Next, θ is the tilt angle from n₀ to the main magnetic field B₀, about which there is cylindrical symmetry. Lastly, θ″ and ϕ″ are the angles for overall transformation from n to B₀ [20].

Fig. 3

Sample geometry is related to nuclear spin interaction tensors in solid-state NMR spectroscopy. (a) Case of random distribution of coupling tensor principal symmetry axes. The ²H NMR spectrum for the two spectral branches of an I=1 spin system is graphed as a function of the reduced frequency ξ_± for the EFG tensor principal axes distributed over the surface of a sphere. The probability distribution p(ξ_±)scales as 1/|cosθ̃|, where θ̃ is the angle between the EFG tensor principal z-axis (assumed axially symmetric) and the static external magnetic field B₀. Weak singularities are evident with intensity maxima at θ̃= 90° (equator) and intensity extending to a minimum at θ̃ = 0° (poles). (b) Case of semi-random distribution of coupling tensor principal symmetry axes. At left the positions of the spectral intensity maxima for an individual spectral branch of the I=1 nucleus are depicted. A static uniaxial distribution is considered in which the coupling tensor z-axes are confined to the rim of a cone. Due to inversion and reflection symmetry only the distribution for a quartersphere is needed. The probability distribution p(ξ_±) scales as 1/|cosθ̃|[cos(θ+θ̃_B) – cosθ̃]^1/2[cosθ̃ – cos(θ–θ_B)]^1/2, where θ is the tilt angle of the alignment axis to the main magnetic field B₀ and θ_B is the bond orientation to the alignment axis. At right an axially symmetric spectrum is obtained for zero tilt; with increasing tilt angle the spectral features correspond to the sum and difference of θ and θ_B. At a sufficiently large tilt angle a further weak singularity becomes evident at θ=90°, as in the case of a random distribution. (c) Example of simulated lineshape for a uniaxial static distribution of methyl groups undergoing 3-fold axial rotation, e.g. in the case of an aligned membrane protein. The two spectral branches of the I=1 ²H nucleus are depicted at bottom with the resultant spectrum at top.

Fig. 4

Angular anisotropy of ²H NMR spectra for bacteriorhodopsin in aligned purple membranes yields retinylidene methyl bond orientations. (a) Experimental ²H NMR tilt series and (b) spectral simulations for C1RC²H₃-labeled bR in dark-adapted sample of aligned purple membranes at −50 °C. All spectra (data as well as simulations) have been scaled such that their integral is the same. Tilt angles are indicated next to the spectra; with a bond orientation of 68.6° used in the simulation.

Fig. 5

Global fitting of ²H NMR spectra to theoretical lineshape for a static uniaxial distribution provides methyl bond orientations and mosaic spread. (a) Dependence of the root mean square deviation (RMSD; arbitrary units) between the experimental data and the global fit on the C13–C²H₃ bond angle and the alignment disorder. Representative data correspond to bR in the M state at −50 °C. (b) Cut of the surface in panel (a) at a mosaic spread of 10°. The deviation between the data and the fit is minimal at a bond angle of 40.3°.

Fig. 6

Carbon numbering for the retinylidene chromophore of bacteriorhodopsin. The chromophore was specifically deuterated in the C13-methyl group. The three isomers are the three structures investigated by solid-state deuterium NMR. (a) The all-trans conformer bR_all-t occurs in the active form of bacteriorhodopsin (light-adapted state). (b) The 13-cis, 15-syn conformer occurs in the dark-adapted state in a 2:1 mixture of bR_13-c and bR_all-t. (c) In the M state the configuration is 13-cis, 15-anti and the Schiff base is deprotonated.

Fig. 7

Angular-dependent ²H NMR spectra for bacteriorhodopsin in light-adapted state show differences versus X-ray crystal structure. (a) Comparison of the ²H NMR tilt series spectra (left) and their best least squares fit (right) for light-adapted bacteriorhodopsin deuterium labeled in the C13-methyl group of the chromophore at −50 °C. The best global fit of the tilt series was obtained with a bond orientation of 34.2° for the C13-C²H₃ bond. The least squares residuals are shown in the left panel. (b) Comparison of the ²H NMR tilt series spectra for the light-adapted state with a simulation using as parameters a bond orientation of 21.6° obtained from X-ray diffraction and a mosaic spread of 10° (right panel). The deviations between data and simulation on the left clearly indicate that the results cannot be fitted with this value for the bond orientation.

Fig. 8

Solid-state ²H NMR spectroscopy of aligned purple membranes yields structure for retinylidene chromophore of bacteriorhodopsin in dark-adapted state. (a) Comparison of the dark-adapted ²H NMR tilt series (data) and their best least squares fit (right) for bR deuterium labeled in the C13-methyl group of the chromophore at −50 °C. The best global fit of the tilt series was obtained with a bond angle of 24.7° for the C13-C²H₃ bond of the 13-cis, 15-syn isomer. For further details of the data analysis see text. The least squares residuals are shown in the left panel. (b) Three-dimensional rendering of two possible orientations of C1R methyl labeled retinal in the bR molecule as derived from ²H NMR orientational constraints. Structure corresponds to all-trans, 15-anti isomer of the dark-adapted state. Only the orientation in (a) is consistent with linear dichroism results; whereas that in (b) differs significantly [7]. Orientation (a) is also consistent with the X-ray crystal structure [142]. Adapted with permission from Ref. [7].

Fig. 9

Difference ²H NMR spectra for bacteriorhodopsin deuterium labeled in the C13–C²H₃ bond indicate conformational variation of retinal cofactor between the M, light-adapted, and dark-adapted states. (Top) Comparison of M and dark-adapted spectra; (middle) comparison of M and light-adapted spectra; and (bottom) comparison of light-adapted and dark-adapted spectra. In each case the first spectrum has the bold black color and the second spectrum the gray color; where the tilt angle is 15°. The differences between the normalized spectra are plotted below each pair of spectra.

Fig. 10

Solid-state ²H NMR spectroscopy reveals conformational changes of retinal chromophore during the bacteriorhodopsin photocycle. (a) Experimental ²H NMR tilt series (left) and the corresponding least squares fit (right) for bR ²H-labeled in the C13-methyl group of retinal and trapped in the M intermediate at −50 °C. The tilt angles are indicated between the spectra and their best fits. All the spectra (data as well as simulations) have been scaled such that their integrals are the same. The residuals between data and fit are also plotted in the left panel. The best global fit of the complete tilt series (shown on the right) was obtained with a bond angle of 40.3° for the C13–C²H₃ bond. (b) Retinal orientation in the M state (13-cis, shaded atoms) and the light-adapted state (all-trans, white atoms) of bR obtained from ²H NMR constraints for the C1R-methyl and C5-methyl bonds. The C5-to-N vector characterizes the molecular long axis and is close to the electronic transition dipole moment in the all-trans conformer. Its inclination with respect to the membrane normal is designated by the angle α_P and has a value of 74° in the ground state and 79° in the M intermediate; this change is largely due to the all-trans to 13-cis isomerization. The C5 atom is assumed to have the same coordinates in both states. Adapted with permission from Ref. [8].

Fig. 11

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 Ref. [77].

Fig. 12

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 vs. 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 Ref. [77].

Fig. 13

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 pre-twisting about C11=C12 double bond. Reproduced with permission from Ref. [77].

Fig. 14

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