Intramolecular interactions that induce helical rearrangement upon rhodopsin activation: light-induced structural changes in metarhodopsin IIa probed by cysteine S-H stretching vibrations

Abstract

Rhodopsin undergoes rearrangements of its transmembrane helices after photon absorption to transfer a light signal to the G-protein transducin. To investigate the mechanism by which rhodopsin adopts the transducin-activating conformation, the local environmental changes in the transmembrane region were probed using the cysteine S-H group, whose stretching frequency is well isolated from the other protein vibrational modes. The S-H stretching modes of cysteine residues introduced into Helix III, which contains several key residues for the helical movements, and of native cysteine residues were measured by Fourier transform infrared spectroscopy. This method was applied to metarhodopsin IIa, a precursor of the transducin-activating state in which the intramolecular interactions are likely to produce a state ready for helical movements. No environmental change was observed near the ionic lock between Arg-135 in Helix III and Glu-247 in Helix VI that maintains the inactive conformation. Rather, the cysteine residues that showed environmental changes were located around the chromophore, Ala-164, His-211, and Phe-261. These findings imply that the hydrogen bond between Helix III and Helix V involving Glu-122 and His-211 and the hydrophobic packing between Helix III and Helix VI involving Gly-121, Leu-125, Phe-261, and Trp-265 are altered before the helical rearrangement leading toward the active conformation.

Keywords: Fourier Transform IR (FTIR); G-protein-coupled Receptor (GPCR); Phototransduction; Protein Conformation; Rhodopsin; Vision.

SCHEME 1.

Meta-II equilibrium mixture composition.

FIGURE 1.

Secondary structural model of bovine rhodopsin. Among the 10 native cysteine residues, 6 cysteine residues, which have free S-H groups (red), were replaced by serine residues (C140S, C167S, C185S, C222S, C264S, and C316S). Cys-110 and Cys-187, which form a disulfide bond (blue), were replaced by alanine residues (C110A/C187A). Alternatively, a cysteine residue was introduced into Helix III (A117C, T118C, E122C, I123C, A124C, L125C, W126C, S127C, L128C, V129C, V130C, L131C, A132C, I133C, and E134C) (green).

FIGURE 2.

FTIR spectra of metarhodopsins. Meta-II_aH⁺/Rho spectrum obtained by irradiation at 280 K (b) is compared with Meta-I/Rho (a) and Meta-II_bH⁺/Rho (c) spectra. Meta-I/Rho and Meta-II_bH⁺/Rho spectra were recorded using bovine ROS by irradiation at 240 and 290 K, respectively. Meta-II_aH⁺/Rho spectra of E122C (d) and E134C (e) are superimposed on that of wild type (cyan lines). The double difference spectra (wild-type spectra minus mutant spectra) are shown below (blue lines).

FIGURE 3.

The difference FTIR spectra before and after irradiation at 280 K. Curve 1, recombinant wild-type rhodopsin in PC liposome hydrated with 1 μl of water (Meta-II_aH⁺/Rho spectrum). Curve 2, linear combination of 0.78 Meta-I/Rho and 0.22 Meta-II_bH⁺/Rho spectra. Curve 3, ROS hydrated with 1 μl of water. Curve 4, ROS hydrated with 1 μl of 60% glycerol, 40% water. Curve 5, ROS hydrated with 1 μl of 80% glycerol, 20% water.

FIGURE 4.

Meta-II_aH⁺/Rho spectra of wild type and mutants in the 1800 to 800 cm⁻¹ region. Spectra were obtained by irradiation with >520-nm light at 280 K. The absorption maxima in the visible region are shown in parentheses. The vibrational bands shifted from those of wild type are indicated by triangles.

FIGURE 5.

Meta-II_aH⁺/Rho spectra in the S-H stretching region. Meta-II_aH⁺/Rho spectra for mutants (red lines) are superimposed on that of wild type (cyan lines). The double difference spectra are shown below (blue lines). Scale bar, 1 × 10⁻⁴. Left, Meta-II_aH⁺/Rho spectra for the cysteine-substituted mutants (a–g). Double difference spectra were calculated by subtracting mutant spectra from wild-type spectra. Middle and right, Meta-II_aH⁺/Rho spectra for cysteine-introduced mutants (h–v). Double difference spectra were calculated by subtracting wild-type spectra from mutant spectra. The typical frequency of the S-H stretching mode is shown by the thick bar at the bottom of each panel.

FIGURE 6.

Mapping of cysteine residues that showed environmental changes. The positions of cysteine residues that showed a frequency shift in Meta-II_aH⁺/Rho spectra are indicated in red, those with only intensity change are shown in pink, and those with no change are shown in white. The amino acid side chains are shown by spheres positioned at the β-carbons of the original amino acid residues. The crystal structure of lumirhodopsin (Protein Data Bank code 2HPY) was used as a template.