CONDENSED-MATTER SPECTROSCOPY SPECTRAL METHODS FOR STUDY OF THE G-PROTEIN-COUPLED RECEPTOR RHODOPSIN. II. MAGNETIC RESONANCE METHODS

Abstract

This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state ²H and ¹³C NMR methods yield information about the both local structure and dynamics of the cofactor bound to the protein and its light induced changes. Complementary site-directed spin labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multi-scale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

Fig. 1

(A) Temperature dependence of the experimental (solid lines) and theoretical (dotted line) solid-state ²H NMR spectra of selectively ²H-labeled C5-, C9-, and C13-methyl groups of the retinylidene chromophore bound to rhodopsin in the dark state [15]. Rhodopsin was recombined with POPC lipid membranes, which were then aligned on the glass slides. The central narrow peak observed at T = −30 °C is due to the natural abundance of deuterium in water. (B) The carbon numbering scheme and retinal structures in dark state of rhodopsin obtained from ²H NMR spectra for the simplified three-plane model [8]. (C) The extended (with rotation around the double bond C11=C12) three-plane model is adopted for analysis of the retinal conformation and orientation within the rhodopsin binding pocket [9, 15]. Figure is taken from Ref. [15].

Fig. 2

Solid-state ¹³C NMR spectra of the vinyl region obtained with cross-polarization and magic angle spinning for rhodopsin regenerated with retinal selectively ¹³C-labeled at (A) positions C10 and C20, and (B) C11 and C20 [2]. The spectra were measured for the rotational resonance condition n = 1, with the rotation speed 11794 ± 3 kHz and 12592 ± 3 kHz, respectively at a resonance frequency of 100.6 MHz. Right panels show the second derivative of the signal for (C) 11-Z-[10,20-¹³C₂]- and (D) 11-Z-[11,20-¹³C₂]-retinylidene rhodopsin in the vinyl region. Figure is adapted from Ref. [2].

Fig. 3

Dipolar-assisted rotational-resonance (DARR) solid-state ¹³C NMR reveals contacts of retinal ¹³C16 and ¹³C17 with phenylalanine, histidine, and methionine residues and changes in the structure of rhodopsin upon light activation. (A) Difference ¹³C NMR spectrum of rhodopsin minus Meta-II showing the region of the ¹³C16 and ¹³C17 resonances of the retinal. (B) Section taken from the 2D DARR ¹³C NMR spectra of rhodopsin containing ¹³C-ring-labeled phenylalanine. The section passes through the diagonal of the ¹³C resonances of the phenylalanine ring, showing only the area of the cross-peaks of the retinal ¹³C16 and ¹³C17 resonances. (C) Similar spectrum in the same region as in (B) in the Meta-II state. (D, E) Representative 2D ¹³C DARR NMR spectra for the dark (black) and Meta-II (gray) states of rhodopsin ¹³C-labeled at position C1 of (D) histidine and (E) methionine and regenerated with 11-Z-[16,17-¹³C₂]-retinal. (F) Structure of rhodopsin in dark state (Protein Data Bank code 1U19). The most significant changes of the configuration of the ligand, the side chains of amino acid residues, and the helices of the protein are indicated by arrows. Figure is adapted from Ref. [7].

Fig. 4

(A) The temperature dependences of the ²H NMR relaxation times of Zeeman (T_1Z, filled symbols) and quadrupolar (T_1Q, empty symbols) order of selectively deuterated C5-, C9- and C13-methyl groups of retinal chromophore in the dark state. Theoretical dependences are shown in solid (T_1Z) and dashed (T_1Q) lines and were calculated for a model of continuous rotational diffusion with coefficients D_|| and D_⊥ (for the case of D_⊥ = 0), and for a model of random rotational jumps about the three-fold symmetry axis with jump rate k between the energy minima. (B) Schematic representation of rhodopsin with bound retinal in the dark state and rotational transformations characterized by the Euler angles Ω_ij(t) used in theoretical analysis of molecular mobility of deuterated methyl groups. The coordinate systems describe fluctuating orientations of the tensor of the quadrupolar interactions of the deuterium nuclei. Here PAS denotes the principal axis system of the tensor of the electric field gradient for the deuterium nucleus (z-axis is parallel to the C–D bond), M is the coordinate system associated with a methyl group (z-axis is parallel to the axis of symmetry and C–CD₃ bond), D is the coordinate system associated with the membrane (z-axis is parallel to the membrane normal n₀), and finally L is laboratory coordinate system (z-axis is parallel to the magnetic field of the NMR spectrometer B₀). Figure is adapted from Ref. [14].