High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Abstract

Site-directed spin labeling has qualitatively shown that a key event during activation of rhodopsin is a rigid-body movement of transmembrane helix 6 (TM6) at the cytoplasmic surface of the molecule. To place this result on a quantitative footing, and to identify movements of other helices upon photoactivation, double electron-electron resonance (DEER) spectroscopy was used to determine distances and distance changes between pairs of nitroxide side chains introduced in helices at the cytoplasmic surface of rhodopsin. Sixteen pairs were selected from a set of nine individual sites, each located on the solvent exposed surface of the protein where structural perturbation due to the presence of the label is minimized. Importantly, the EPR spectra of the labeled proteins change little or not at all upon photoactivation, suggesting that rigid-body motions of helices rather than rearrangement of the nitroxide side chains are responsible for observed distance changes. For inactive rhodopsin, it was possible to find a globally minimized arrangement of nitroxide locations that simultaneously satisfied the crystal structure of rhodopsin (Protein Data Bank entry 1GZM), the experimentally measured distance data, and the known rotamers of the nitroxide side chain. A similar analysis of the data for activated rhodopsin yielded a new geometry consistent with a 5-A outward movement of TM6 and smaller movements involving TM1, TM7, and the C-terminal sequence following helix H8. The positions of nitroxides in other helices at the cytoplasmic surface remained largely unchanged.

Fig. 1.

View of the cytoplasmic face of inactive rhodopsin (PDB entry 1GZM) showing modeled R1 side chains for all sites studied here. The dotted lines connect pairs where the interspin distance distribution was determined by using DEER. Each distribution was measured before and after light-activation. The R1 side chains are modeled from known conformations (see Methods). (Inset) Structure of R1 with the dihedral angles defined.

Fig. 2.

Representative selection of DEER results in the R (solid line) and R* states (dotted line). (a) Normalized dipolar evolution after removal of the exponential background. The high-frequency signal is due to proton modulations (28). (b) DC centered Fourier transform of the data in a. (c) Distance distribution calculated from the data in b by using Tikhonov regularization.

Fig. 3.

Projection contours of the spin locations calculated from the measured distance distributions for representative sites (see text for details). (a) Probable locations of the nitroxide spins in R shown in a gradient from blue to purple and overlaid on a ribbon model of R from the crystal structure (PDB entry 1GZM). (b) Corresponding locations in R* shown in a gradient from red to yellow, and, for reference, the ribbon model and typical contours (dotted traces) for R overlaid. The center used to obtain the data in Fig. 4 is shown.

Fig. 4.

After defining a central reference point halfway between the dark location of 74 and 252 (see Fig. 3b), the radial distribution profiles along a line connecting the center and the most probable location is calculated for each site. Each trace incorporates data from two to four measured interspin distance distributions.