Rhodopsin disulfide cross-linking

Cai, K. Klein-Seetharaman, J. Hwa, J. Hubbell, W.L. Khorana, H.G. Structure and function in rhodopsin Effects of disulfide cross-links in the cyto-... 

Hubbell, W. L., Altenbach, C., Hubbell, C. M., and Khorana, H. G. (2003). Rhodopsin structure, dynamics, and activation A perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking. Adv. Protein Chem. 63, 243-290. 

Cysteine-scanning mutagenesis, involving more than 100 mutations, has been systematically carried out through Cl—C3, the cytoplasmic terminations of TM1-TM7, H8, and the C-terminal tail. In addition, more than 40 pairs of cysteines have been introduced at the cytoplasmic face. With these mutants as a basis set, three classes of experiments have been carried out, namely SDSL, sulfhydryl reactivity, and disulfide cross-linking kinetics. A global comparison of the results provides a unique view of the solution state, its dynamics, and its correlation with the crystal structure. By solution state is meant, in all cases, rhodopsin solubilized in dodecyl maltoside (DM) micelles. The measured functional properties of rhodopsin, namely transducin activation (Resek et al., 1993) and phosphorylation by the rhodopsin kinase (Thurmond et al., 1997), are conserved in this detergent, and it is presumed to be a reasonable approximation to the bilayer environment. 

A. Overview of Methods for Exploring the Solution Structure of Rhodopsin Site-Directed Spin Labeling Sulfhydryl Reactivity, and Disulfide Cross-Linking... 

Fig. 9. Proximity relationships for residues in Cl relative to 316 in H8. Example of R1 distance mapping in rhodopsin. For each distance measurement, only two R1 side chains were in the protein, one fixed at the reference site 316, and the other at a site in the sequence 55-75. The R1 side chains were modeled based on crystal structure data with energy minimization subject to the experimentally determined distance constraint (shown). In each case, the measured distances in solution were in good agreement with those expected from the rhodopsin crystal structure. Substituted cysteine residues 65 and 68 most rapidly formed disulfide cross-links with the reference cysteine at 316 in H8. This is indicated by the dark bars connecting the potential disulfide partners.

Twelve cross-links designed to explore the role of helix movement at the cytoplasmic surface of rhodopsin are shown in Fig. 22. An important conclusion from these studies is that any interhelical cross-link involving TM6 blocks activation. These include the five disulfide cross-links between 139C in TM3 and a second cysteine in the sequence 247C-251C in TM6 (Farrens et al., 1996 Cai et al., 1999b), the cross-link between... 

As described in the previous sections, cysteine scanning mutagenesis and the associated techniques of SDSL, sulfhydryl reactivity, and disulfide cross-linking rates have provided a rather detailed view of rhodopsin dynamics in solution and conformational changes leading to the activated state. In this section, the structural origins of these functional properties in solution are examined from the point of view of the crystal structure. 

The focus of this review has been on rhodopsin structure and dynamics at the cytoplasmic face in solution, the comparison between the solution structure and the crystal structure, and the structural changes underlying receptor activation. The data from SDSL and disulfide cross-linking together indicate that the crystal and solution structures are very similar at the level of the backbone fold for Cl, H8, and the cytoplasmic termination of TM1-TM7. However, substantial differences are seen in C3 and the C-terminal tail, wherein the backbones are flexible on the nanosecond time scale in solution. Not surprisingly, these are the most disordered regions in the crystal lattice and correspond to sequences important for function. 

Fig. 22. Cross-links introduced into rhodopsin to test role of helix movement in activation. Of the 12 cross-links shown, 11 are disulfide bonds produced by oxidation of engineered cysteine pairs.The pairs are 139/247 (i), 139/248(i), 139/249(i), 139/250(i), 139/251 (i), 65/316(p), 136/222 (i), 136/225(p), 140-222 (i), 140/225(p). One cross-link, 138/251 (i) was produced by Zn2+ binding (see text). The letter in parentheses following each pair identifies the cross-link as inhibitory (i) or permissive (p) with respect to transducin activation. Inhibitory bonds are shown in black, and permissive in gray.

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