PMC

Reaction of the methanethiosulfonate spin-labeling reagent with cysteine to generate the nitroxide side chain, R1. The dihedral angles (X₁-X₅) are defined.

EPR spectra of 140R1, 227R1, 250R1, and 316R1 in digitonin (red) compared with those in DM (green) and PC:PE:PS bilayers (blue). In (A)-(D), spectra of the indicated spin-labeled mutant in R and R* are compared for the micellar and bilayer hosts. The R* - R difference spectrum in digitonin is shown in part [3].

The inactive (R, MI, and Op) and active (MII* and Op*) states of rhodopsin in native membranes. The retinal isomer and protonation state of the Schiff base linkage (PSB⁺ = protonated and SB⁰ = unprotonated) are noted under each species. The unliganded opsin (Op) is inactive but may be activated either by lowering the pH or by constitutively active mutations (CAM’s).

Example of light - dark difference spectra (black trace) of the labeled rhodopsin mutants in DM and PC:PE:PS bilayers at pH 6 and 20 °C. The fit to the difference spectrum (dashed orange trace) and the contributions of R (blue trace), MI (green trace), and MII (red trace) to the difference spectrum are also shown. The magenta trace is the residual from the fit.

Ribbon model of rhodopsin in the inactive state (PDB ID 1GZM). Yellow spheres show the C_α atoms corresponding to sites where the R1 side chain was substituted in the present study (140, 227, 250, and 316). The location of the lipid bilayer relative to the protein was positioned according to SDSL and electron microscopy studies (see text). Helices are color coded as follows: TM3, green; TM5, blue; TM6, red; H8, magenta.

Simulated EPR spectra and semiempirical parameters used to infer nitroxide mobility for different dynamic modes. (A) Rapid anisotropic motion. (B) Slow isotropic motion. (C) A complex state corresponding to a weighted sum of (A) and (B). The central line width (ΔH₀) and splitting of resolved hyperfine extrema (2A_zz’) are identified. Spectral intensity in regions α and β identifies relatively immobile and mobile components, respectively.

EPR spectra of rhodopsin 227R1 in DM and in PC:PE:PS bilayers at pH 6, 20 °C, and a molecular model of the side chain. (A) Spectra for the indicated samples (parts [1] and [2]). R* - R difference spectra are shown to highlight the changes upon light activation (parts [3] and [4]). (B) A model of one rotamer of 227R1in the 1GZM rhodopsin crystal structure. TM5 is shown in blue, and nearby phospholipids are modeled.

EPR spectra of 316R1 in DM and PC:PE:PS bilayers at pH 6, 20 °C, and a molecular model of the side chain. (A) Spectra of the indicated samples (parts [1] and [2]). R* - R difference spectra are shown to highlight the changes upon light activation (parts [3] and [4]). (B) A model of 316R1 in the 1GZM rhodopsin crystal structure. Helix H8 is shown in magenta. For clarity, the cytoplasmic end of TM1 and the loop between TM1 and TM2 are shown as gray ribbons.

pH dependence of the EPR spectra of 250R1 in DM and PC:PE:PS bilayers at 20 °C. (A) Spectra for the indicated samples in DM (parts [1] and [2]). (B) Direct comparison of the spectra for R and R* in DM at pH 6 and 8. (C) Spectra for the corresponding samples in PC:PE:PS bilayers (parts [1] and [2]). In (A) and (C), R* - R difference spectra are shown to highlight the changes upon light activation at pH 6 and 8 (parts [3] and [4]). The insets for the EPR spectra show more clearly the differences in the low and high fields.

EPR spectra of rhodopsin 140R1 in DM and PC:PE:PS bilayers at pH 6 and molecular models of the side chain. (A) Spectra for the indicated samples at 20 °C (parts [1] and [2]). (B) Spectra for the indicated samples at 35 °C (parts [1] and [2]). In (A) and (B), R* - R difference spectra are shown to highlight the changes upon light activation (parts [3] and [4]). (C) Models of 140R1 in the 1GZM rhodopsin crystal structure. TM3 is shown in green, a portion of TM5 is shown as a blue ribbon, and a nearby phospholipid is modeled.

Experimental and simulated EPR spectra of rhodopsin 250R1 in DM and PC:PE:PS bilayers at pH 6, 20 °C, and a molecular model of the side chain. (A) Experimental spectra for the indicated samples (parts [1] and [2]). R* - R difference spectra are shown to highlight the changes upon light activation (parts [3] and [4]). (B) Simulated spectra (red dotted traces) of 250R1 R and R* in DM and bilayers are shown superimposed on the corresponding experimental spectra (solid blue traces). (C) Space-filling model of 250R1 (carbon atoms shown in green) in the 1GZM rhodopsin crystal structure viewed from the cytoplasmic surface.