Sulfhydryl reactivity, rhodopsin

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. 

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

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. 

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. 

Studies of TM residue accessibility to water-soluble sulfhydryl-reactive compounds has allowed Javitch and coworkers to gain evidence that P2-AR activation also involves a conformational rearrangement of TM VI (123), whereas detailed fluorescence spectroscopy studies by Kobilka and coworkers indicated that the helical movements occurring with P2-AR activation are almost identical to those for rhodopsin, that is, a counterclockwise rotation (when viewed from the extracellular surface of the receptor) of both TM III and TM VI, with a tilting of the cytoplasmic end of the latter toward TM V (74,124). The importance of the orientation of TM VI, which is stabilized by interhelical interactions with TM V, comes from studies of the arARs, which showed that mutation of either the... 

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