Accompanied rigid-body movements of TM3 and TM6 [46] expose a hydrophobic cleft between TM5 and TM6 [47]. Exposure of this hydrophobic site draws a nearby phenylalanine (F64) of the Gtγ, an orientation otherwise unfavored in an aqueous environment, resulting in amphipathic helix formation of the Gtγ-C-terminus, stabilized by activated rhodopsin, allowing allosteric regulation of nucleotide exchange [48].

STRUCTURAL ANALYSIS

The current state of structural and functional knowledge regarding rhodopsin has been recently reviewed [49, 50]. Rhodopsin is ellipsoid in shape with dimensions of approximately 75 × 48 × 35 Å. The 348 amino acids of bovine rhodopsin are posttranslationally modified with N-terminus acetylation, N-terminus dual glycosylations (at N2 and N15), an intradiskal disulfide bond (C110–C187), dual palmitoylations at the C-terminus (C322 and C323), and multiple C-terminus light-activated phosphorylations.

Electron Cryomicroscopy and Crystal Structure

Most structural information on rhodopsin, and thereby on GPCRs, is based on an inactive, inverse-agonist-bound, dark state. This is because the most definitive structural data available are from x-ray crystallography. Electron cryomicroscopy leveraged twodimensional crystal formations to provide the earliest, albeit low-resolution, pictures of rhodopsin structure [16]. Originally solved to 2.8 Å [20], the crystal structure of rhodopsin demonstrated a number of important principles in rhodopsin function, the structure of GPCRs, and general aspects of large integral membrane proteins. Importantly, as mentioned, the helical arrangement was shown to be significantly different from, and more organized than, bacterial rhodopsin (another intensely studied seven- transmembrane-spanning protein that serves as a proton pump from halophilic archaebacteria). Identification of proline-induced bending of transmembrane helices (e.g., highly conserved P267) showed significant distortion from an ideal helix, facilitating interhelical interactions and allowing for chromophore accommodation. Of particular interest was identification of a water-mediated interhelical interaction network centered around Asp83 on TM2, connecting this helix to TM3 and TM7 through interactions with Gly120 and Asn302, respectively. Also of mechanistic importance was the structural suggestion that β-ionone movement toward TM3 may result in helical displacement.

Additional crystal analyses improved structural detail by adding missing residues (protein databank (http://rscb.org) structure identification numbers (pdf:1HZX), increasing resolution to 2.6Å (1L9H) [51] (Fig. 3B), further improving resolution to 2.2 Å (1U19) [52], and refining earlier structures in different crystal space groups (1GZM). Increased resolving power provided confirmation of the structural importance of water molecules and their likely participation in spectral tuning and proton transfer [51]. Further details provided definition of the cytoplasmic region and chromophore, demonstrating a 6 s-cis conformation of the ionone ring and an unusual twisted and extended π-system with a delocalized charge–carboxylate interaction [52]. Also clarified was the hydrogen-bond- ing network connecting E113 to E181 through a required water molecule, a network later confirmed to transmit the counterion shift important in rhodopsin activation [45].

Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) resolution of rhodopsin domain peptides have also been used to study the structure of rhodopsin [53]. Nuclear magnetic resonance has also extended crystallographic interpretations toward the elucidation of structural changes involved in opsin activation [54, 55]. Solid-state NMR, in particular, providing advantages for dealing with integral membrane proteins, is uniquely adept for following particular interactions, with studies utilizing isotopically labeled retinal providing direct chemical observation of modifications to ligand–protein interactions following light activation [55, 56]. The complete 1H and 13C assignments of the chromophore in the bound state showed interactions between 11-cis retinylidene’s H16/H17 and Phe 208, Phe 212, and H18 with Trp265 [57]. NMR provides considerable advantages in the context of activation as light-activated rhodopsin remains elusive to crystallization, and photoactivated intermediates, likely due to their transient nature, prove similarly difficult to crystallize [58]. The side chain of Glu122 and backbone of His211 were shown to be disrupted in meta II [55].

Cysteine Mutagenesis and Electron Paramagnetic Resonance

Site-directed cysteine mutagenesis and sulfhydryl modification chemistry provide remarkable resources for structural studies facilitating spin labeling, disulfide construction [18, 59], and metal-binding site engineering [19]. Paramagnetic spin labeling adds unique topological information, measuring solvent accessibility of the modified residue, overall mobility of particular residues, and even demonstrating residue interactions. A series of spin-labeling studies revealed structural details of cytoplasmic loop 1 connecting helices 1 and 2 [60], loop 2 between helices 3 and 4 [12], loop 3 between helices 5 and 6 [61, 62], loop 4 leading from helix 7 to the palmitoylation site [63], and the C-terminal tail [64]. Together, these results map a range of light-initiated structural changes. Evaluating light-activated changes in mobility of cytoplasmic loop 3, the transducin-interacting domain connecting transmembrane helices 5 and 6, demonstrated a dramatic loss of mobility for residue V227, with a smaller decrease in mobility for K231. The loss of mobility corresponds to formation of tertiary contacts, whereas an increased mobility, as observed with V250, T251, M253, and Q244, indicates that tertiary contacts are lost in conversion to MII. This has considerable mechanistic importance as Q244 has been identified as a required residue for Gt activation [65]. As such, it can be seen that residues in cytoplasmic loop 3 are exposed to allow transducin interaction with nearby residues, forming new contacts to maintain structural integrity of the receptor.

Other Approaches

Breaking the protein into subsections for structural analysis of the components attempts to alleviate some of these concerns; however, less-direct biophysical measurements of intact protein have allowed structural inferences to fill gaps left by direct measurements. Fourier-transform infrared (FTIR) spectroscopy, for example, resolved proton movements involved in activation-induced counterion shift [44], and ultraviolet-visible (UV/Vis) spectrophotometry is routinely used to evaluate rhodopsin purity, structural stability, regeneration rate, and activation state [66]. Such techniques prove quite powerful

at leveraging structural information provided by crystallographic data, particularly when combined with complementary tools such as site-directed mutagenesis and molecular modeling. In fact, molecular modeling has been pivotal in the study of rhodopsin, as it is a critical refining step in processing crystallographic and NMR data, and facilitates mutagenic approaches and biophysical data interpretation. Not surprisingly, as modeling techniques continue to mature, they become utilized as an experimental approach in their own right, with energy-induced decay of the protein structure revealing a core set of stabilizing interactions providing a folding scaffold for the overall rhodopsin structure [67].

Genetic manipulation techniques have proven useful in structural investigations of rhodopsin. From deletion of segments and chimeric recombination of protein to mutagenesis of individual residues and chemical modification of localized functional groups, each distinct application provides creative insight into structural features of this remarkable protein. Supporting a common GPCR activation principal is construction of a chimeric rhodopsin spliced with the cytoplasmic regions of the β-adrenergic receptor, resulting in a light-activated GPCR that elicits a β-adrenergic Gαs stimulation of cyclic adenosine monophosphate (cAMP) [68].

Investigations into rhodopsin structure and function parallel many major unanswered questions facing general protein biochemistry. Structural motions involved in translating binding of a ligand or allosteric modulator across the membrane bilayer to activate intracellular signals are of general interest, particularly if findings extrapolate to a wider range of GPCRs. More structurally accessible than most GPCRs, rhodopsin continues to provide a uniquely suited prototype for studying general GPCR features. One interesting feature to develop over recent years is the concept of GPCR dimerization, carrying uncertain potential impact [69, 70]. Rhodopsin has demonstrated potential to form dimers, as well as higher-order oligomers, in disk membranes [33], expression systems [71], liposomes [72] and in solubilizing detergents [34]. However, demonstration of native receptor dimerization, along with a functional requirement for dimerization, remains elusive. Perhaps the most compelling evidence in favor of rhodopsin dimer formation was demonstrated using atomic force microscopy [30]. A large battery of additional techniques, including electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, proteolysis, and cross-linking support the idea that rhodopsin is capable of dimer formation in both isolated disk membranes and when solubilized in detergent [33]. Site-directed mutagenesis, combined with fluorescence resonance energy transfer (FRET) and cysteine cross-linking, suggests a hydrophobic interaction between W175 in the second intradiskal loop, and Y206 in TM5 participates in rhodopsin dimer formation [71]. Molecular modeling has provided additional details for the putative interface [31].

Implications of native dimer formation for GPCRs range from transport considerations [73] to activation responses [74]. An interesting approach using solubilized rhodopsin in detergent micelles of increasing size accommodating mixtures of differing oligomeric sizes suggested that larger dimeric organizations might be structurally preferred, reflected by increased receptor stability [34]. Although these organizations may not be required for transducin activation, increasing levels of oligomerization corresponded to dramatically increased rates of Gt activation, without modifying MII decay, consistent with putatively improved Gt binding by dimeric rhodopsin [75].