1 Introduction Organisms of all domains of life use photoreceptor proteins to sense and respond to light. The light-sensitivity of photoreceptor proteins arises from bound chromophores such as retinal in retinylidene proteins, bilin in biliproteins, and flavin in flavoproteins. Rhodopsins found in Eukaryotes, Bacteria, and Archaea consist of opsin apoproteins and a covalently linked retinal which is employed to absorb photons for energy conversion or the initiation of intra- or intercellular signaling. 1 Both functions are important for organisms to survive and to adapt to the environment. While lower organisms utilize the family of microbial rhodopsins for both purposes, animals solely use a different family of rhodopsins, a specialized subset of G-protein-coupled receptors (GPCRs). 1,2 Animal rhodopsins, for example, are employed in visual and nonvisual phototransduction, in the maintenance of the circadian clock and as photoisomerases. 3,4 While sharing practically no sequence similarity, microbial and animal rhodopsins, also termed type-I and type-II rhodopsins, respectively, share a common architecture of seven transmembrane α-helices (TM) with the N- and C-terminus facing out- and inside of the cell, respectively (Figure 1). 1,5 Retinal is attached by a Schiff base linkage to the ε-amino group of a lysine side chain in the middle of TM7 (Figures 1 and 2). The retinal Schiff base (RSB) is protonated (RSBH+) in most cases, and changes in protonation state are integral to the signaling or transport activity of rhodopsins. Figure 1 Topology of the retinal proteins. (A) These membrane proteins contain seven α-helices (typically denoted helix A to G in microbial opsins and TM1 to 7 in the animal opsins) spanning the lipid bilayer. The N-terminus faces the outside of the cell and the C-terminus the inside. Retinal is covalently attached to a lysine side chain on helix G or TM7, respectively. (B) Cartoon representation of the helical arrangement of a microbial rhodopsin with attached all-trans-retinal (bacteriorhodopsin, PDB ID: 1C3W). Figure 2 Genesis of the chromophore of microbial and animal rhodopsins. Cleavage of β-carotene is the source of the chromophore. The ground state of microbial and animal rhodopsins possesses all-trans- and 11-cis-retinal as its chromophore, respectively, bound to a Lys residue via a Schiff base, which is normally protonated and exists in the 15-anti configuration. It should be noted that microbial rhodopsins depend exclusively on all-trans-retinal, while some animal rhodopsins possess vitamin A2 (C3=C4 double bond for fish visual pigments) and hydroxyl (C3—OH for insect visual pigments) forms of 11-cis-retinal. Usually, photoactivation isomerizes microbial rhodopsin selectively at the C13=C14 double bond and animal rhodopsin at the C11=C12 double bond. Retinal, the aldehyde of vitamin A, is derived from β-carotene and is utilized in the all-trans/13-cis configurations in microbial rhodopsins and the 11-cis/all-trans configurations in animal rhodopsins (Figure 2). 1,6 For optimal light to energy or light to signal conversion, defined chromophore–protein interactions in rhodopsins direct the unique photophysical and photochemical processes, which start with specific retinal isomerization and culminate with distinct protein conformational changes. The protein environment is typically optimized for light-induced retinal isomerization from all-trans → 13-cis in microbial rhodopsins and for 11-cis → all-trans in animal rhodopsins. Variations in this isomerization pattern are discussed in sections 3 and 4. The 7TM protein scaffold of microbial rhodopsins is designed for light-driven ion pumps, light-gated ion channels, and light sensors which couple to transducer proteins (Figure 3). 7 Microbial rhodopsins were first found in the Archaea (Halobacterium salinarum, historically referred to as Halobacterium halobium) 8 and were therefore initially termed archaeal rhodopsins. H. salinarum contains bacteriorhodopsin (BR) 8 and halorhodopsin (HR) 9 that act as a light-driven outward proton pump and inward chloride pump, respectively. As ion pumps, they contribute to the formation of a membrane potential and thus have their function in light–energy conversion. The other two H. salinarum rhodopsins are sensory rhodopsin I and II (SRI and SRII), 10 which act as positive and negative phototaxis sensors, respectively. Since the original discovery of BR in H. salinarum, similar rhodopsins have been found in Eubacteria and lower Eukaryota, leading to the name microbial rhodopsins. For example, Anabaena sensory rhodopsin (ASR), the first sensory rhodopsin observed in the Eubacteria, 11 is a sensor that activates a soluble transducer (Figure 3). Figure 3 Microbial rhodopsins can function as pumps, channels, and light-sensors. Arrows indicate the direction of transport or flow of signal: (A) light-driven inward chloride pump (halorhodopsin (HR), PDB ID: 1E12), (B) light-driven outward proton pump (bacteriorhodopsin (BR), PDB ID: 1C3W), (C) light-gated cation channel (channelrhodopsin (ChR), PDB ID: 3UG9), (D) light-sensor activating transmembrane transducer protein (sensory rhodopsin II (SRII), PDB ID: 1JGJ), (E) light-sensor activating soluble transducer protein (Anabaena sensory rhodopsin (ASR), PDB ID: 1XIO). Channelrhodopsins (ChRs), another group of microbial rhodopsins, were discovered in green algae where they function as light-gated cation channels within the algal eye to depolarize the plasma membrane upon light absorption. 12,13 The primary depolarization of the eyespot membrane is transferred to the flagellar membrane and results in a reorientation of the alga toward a light source (photophobic responses and phototaxis). Thus, ChRs naturally function as signaling photoreceptors as well. Discovery of ChR led to an emergence of a new field, optogenetics, 14 in which light-gated ion channels and light-driven ion pumps are used to depolarize and hyperpolarize selected cells of neuronal networks, e.g., for therapeutic reasons 15 or in order to understand the circuitry of the brain. 16,17 Thus, studies on microbial rhodopsins are beneficial not only for our basic understanding of retinal proteins, but also for providing a toolset to study neuronal signaling through optogenetics. Animal rhodopsins belong to the superfamily of GPCRs which detect extracellular signals, typically by binding small molecule ligands like hormones and neurotransmitters. 18,19 By a ligand-induced conformational change, GPCRs become activated and capable of transducing the activation signal by catalyzing GDP/GTP exchange on membrane-bound heterotrimeric G proteins within the cell, thus initiating G protein-mediated signaling cascades (Figure 4). 19 After activation-dependent phosphorylation by a G-protein-coupled receptor kinase, active GPCRs can also interact with arrestin to effect G protein-independent signaling and attenuation of the ligand-mediated activation signal. 20 Animal rhodopsins are typically specialized GPCRs, capable of detecting single photons as a physical stimulus. 21 Because the 11-cis-retinal ligand is covalently bound within the retinal-binding pocket of the receptor, photon absorption and the ensuing retinal cis → trans isomerization convert an inactivating ligand (the inverse agonist 11-cis-retinal) into an activating ligand (the agonist all-trans-retinal) in situ. Vertebrate rhodopsin was discovered more than 130 years ago and has long been used as a prototypical GPCR. 22 Due to the relative ease of purification from native material, it has been studied extensively. 2 Figure 4 Animal rhodopsins are specialized G-protein-coupled receptors (GPCRs). (A) Binding of extracellular ligands stabilizes certain GPCR conformations which enable the GPCR to catalyze GDP/GTP exchange in heterotrimeric G proteins (Gαβγ) and/or to induce G-protein-independent, arrestin-mediated signaling. (B) Typical GPCR fold shown in cartoon representation for bovine rhodopsin (PDB ID: 1U19). Structures of animal and microbial rhodopsins differ largely (cf. Figure 1B) and are drawn in opposite orientations with respect to the membrane. As model for the GPCR family, animal rhodopsin is shown in the orientation commonly used for GPCRs. In a large number of publications, animal rhodopsins are shown for historical reasons in the orientation of microbial rhodopsins (C-terminus up). The goal of this review is to provide mechanistic insights from biophysical and structural studies into the function of microbial and animal rhodopsins, with the latter as representatives of GPCRs. After a general description of retinal photoisomerization in section 2, the functional mechanisms of various basic types of microbial rhodopsins will be discussed in section 3. In section 4, animal rhodopsins will be reviewed with a focus on bovine visual rhodopsin (abbreviated as Rho in some cases), the photoreceptor in retinal rod cells, followed by some extension to color visual rhodopsins and invertebrate rhodopsins. 2 Light Absorption and Photoisomerization 2.1 Color Tuning Light absorption initiates functions of both microbial and animal rhodopsins, 23,24 and the wavelength dependence of the absorption efficiency determines the colors of the proteins (Figure 5). The length of the π-conjugated polyene chain in the retinal chromophore as well as the protonation of the RSB linkage determine the energy gap of the π–π* transition, 25 so that the absorption of most rhodopsins is within the visible region (400–700 nm). Humans have a single photoreceptor for dim light vision (rhodopsin, λmax ∼500 nm) and three receptors for color vision (blue, λmax ∼425 nm; green, λmax ∼530 nm; red, λmax ∼560 nm), 26,27 whereas some shrimp species contain up to 16 rhodopsins covering the spectral range from 300 to 700 nm. 28 While the chromophore molecule is usually the same in all pigments (retinal bound via a (protonated) Schiff base), the absorption maxima differ significantly, implying an active protein control of the energy gap between the ground and excited states of the retinal chromophore. The mechanism of color tuning has fascinated researchers for a long time, and several factors have been determined to be responsible for it. Figure 5 Microbial rhodopsins exhibit a wide range of absorption maxima. Colors of microbial rhodopsins (A) and their absorption spectra (B). The following rhodopsins are shown: (1) a blue-proteorhodopsin (LC1-200, pH 7), (2) Q105L mutant of LC1-200 (pH 7), (3) a green-proteorhodopsin (EBAC31A08, pH 7), (4) A178R mutant of green-proteorhodopsin (pH 7), (5) bacteriorhodopsin (pH 7), (6) H. salinarum sensory rhodopsin I (pH 4). The protonation state of the chromophore plays a crucial role in color tuning; the unprotonated RSB absorbs in the UV region (λmax ∼360–380 nm), and this absorption is quite insensitive to the environment in contrast to the RSBH+, which exhibits a large variation in absorption covering the entire visible light spectrum. Other factors defining the spectral tuning of individual rhodopsins are given by chromophore–protein interactions such as electrostatic interactions with charged and polar amino acids, termed electrostatic tuning and extensively studied, first using retinal analogues, 29−32 and, later, site-directed mutagenesis. 33−35 Electrostatic tuning was elegantly demonstrated in a model system based on cellular retinol-binding protein II. This system was engineered to covalently bind all-trans-retinal and its absorption maximum was changed from 425 to 644 nm via mutations that changed the electrostatic potential within the retinal-binding pocket. 36 Interactions of retinal with charged, polar, and aromatic amino acids play a role in changing the electronic energy levels, as do hydrogen-bonding interactions and steric contact effects. Strong hydrogen bonds can lead to charge transfer, and steric contacts can lead to a twist of retinal. All these tuning processes in concert shape the absorbance maxima of retinal in microbial and animal rhodopsins. One of the most prominent factors in color tuning is the interaction of retinal with the counterion(s) (Figure 6). In the ground state, the retinal chromophore is positively charged due to RSBH+ (C=NH+). The excited state has strong charge transfer character where the positive charge is displaced toward the β-ionone ring, leading to a neutralization of the RSBH+ (Figure 6B). 37,38 Interaction of the RSBH+ with the negatively charged counterion(s) in microbial and animal rhodopsins leads to an electrostatic stabilization in the electronic ground state of retinal accompanied by an increase of the RSB pKa (Figure 6A). The resulting larger energy gap between ground and excited states causes a blue-shift of the absorption (Figure 6D, compare cases [A] and [B]). 39,40 If a negative charge is located near the β-ionone ring, the excited state is energetically stabilized compared to the ground state (Figure 6C,D), which leads to a smaller energy gap and therefore to a red-shift of the wavelength of electronic excitation. As the absorption maximum of isolated all-trans RSBH+ in gas phase is 610 nm (Figure 6B,D, case [B]), 40 in principle, absorption in the deep red range (λmax > 600 nm) should be possible for case [C] in Figure 6, while λmax 180 kJ/mol, thermal isomerization of a rhodopsin molecule once every 1010 years would be expected, implying that thermal and light-dependent processes follow different pathways. A more recent theoretical study proposed a pathway of thermal isomerization in rhodopsin with a transition state displaying the same charge-transfer character as the electronically excited state of Rho. 472 From a quantitative relation between rhodopsin’s photoactivation energy and its peak absorption, λmax, it was proposed that dark noise arises from thermal retinal isomerization which needs to overcome the same energy barrier as in the photoisomerization process. 399 On the basis of the slow hydrogen/deuterium exchange of Thr118 in bovine Rho, it was suggested that local protein structural fluctuations transiently widen the retinal binding pocket for thermal retinal isomerization. 473 It is interesting to note that Drosophila rhodopsin also has a light-independent role in temperature discrimination in larvae which may be related to thermal retinal isomerization. 474 To effectively release the structural constraints that stabilize the inactive rhodopsin state, photon energy is absorbed and used for retinal isomerization (cf. section 2), driving subsequent protein conformational changes. About 150 kJ/mol of the initially absorbed photon energy are stored in the “distorted” all-trans-retinal of the Batho photointermediate and gradually dissipated via a transient blue-shifted intermediate (BSI) 475 and the Lumi intermediate, concomitantly with a release of strain in retinal (Figures 21–23), eventually yielding Meta I after a few microseconds. The “early” photointermediates Batho and Lumi can be trapped by low temperature and have been studied structurally, 120,146 as well as spectroscopically. 476−478 The distinct absorption maxima of each intermediate reflect the gradual changes in chromophore–protein interaction. It is not until the formation of Meta I that significant backbone structural changes occur. 479,480 In Batho and to a lesser extent in Lumi intermediates, protein conformational adjustments to retinal relaxation are limited to a few amino acid side chains within the retinal binding pocket. 120,146 When the Meta I state is attained, an equilibrium between Meta I and Meta II states develops (Figure 21), which is dependent upon pH and temperature, with lower pH and higher temperature favoring Meta II. 481,482 Deprotonation of the RSBH+ upon Meta II formation results in the characteristic 100 nm blue-shift of the absorption maximum to the near UV region (λmax = 380 nm). 481,483 Meta II, as defined by its 380 nm absorption, comprises both the isochromic Meta IIa and Meta IIb substates which develop sequentially from Meta I. 484 In Meta IIb, proton uptake occurs to Glu134 of the (D/E)RY motif in TM3, 485,486 explaining why low pH favors Meta II despite the loss of a proton from the RSBH+. 487 Time-resolved EPR studies with bovine Rho in dodecylmaltoside detergent revealed that the large TM6 movement occurs during transition from Meta IIa to Meta IIb and led to the reaction scheme for the Meta states shown in Figure 21. 488 FTIR studies confirmed this reaction scheme for bovine Rho in its native membrane environment. 489,490 Electron crystallography on bovine Rho 2-D crystals that were illuminated and trapped in the Meta I photointermediate by the crystal lattice demonstrated that up until Meta I the protein backbone remains in a conformation similar to that of the rhodopsin dark state. 491 Illumination of 3-D bovine Rho crystals yielded the spectral shift characteristic to Meta II, but only revealed a small TM6 movement and some rearrangement of the cytoplasmic surface. 492,493 On the basis of absorption maximum and the extent of TM6 movement, the structure of this photoactivated Rho most likely represents the Meta IIa state. The Meta IIb state with fully opened cytoplasmic domain is represented in the bovine Meta II structures obtained by reconstitution of Ops* crystals with all-trans-retinal or by illumination of the constitutively active M257Y rhodopsin mutant before crystallization. 426,433 Examination of agonist-bound GPCR structures reveals that GPCRs exist in conformations with differing extents of TM6 movement, with no or little TM6 movement for inverse agonists and larger TM6 movement for partial agonists and full agonists. 418 However, stabilizing mutations, truncations of loops, and in many cases insertion of T4 lysozyme or apocytochrome b562 fusion partners into cytoplasmic loop 3 (connecting TMs 5 and 6) or at the N-terminus have been necessary for the crystallization of all nonrhodopsin GPCRs to have their structures determined to date. 419,494,495 This affects affinity for agonist or antagonist binding and likely exerts some influence upon the degree of movement of TM6. 496−498 NMR and hydrogen/deuterium exchange studies on β2AR lacking fusion partners provide evidence for substantial conformational heterogeneity of agonist- and inverse agonist-bound β2-AR preparations. 499−502 The heterogeneity supports the view of conformational equilibria of GPCRs that can be shifted to either side depending on the type of ligand and are comparable to the Meta I/Meta II equilibrium of rhodopsin (Figures 21 and 22). 503,504 A difference between diffusible ligand-activated GPCRs and the activation of rhodopsin by light is reflected in the mode by which the ligand acts in the activation process. It was proposed that diffusible ligands might select the suitable conformation from the equilibrium of inactive and active GPCR conformations, whereas in an induced-fit scenario the ligand would bind an inactive conformation and induce a conformational change toward the active conformation. 503 Rhodopsin with its activation by retinal photoisomerization is likely to correspond to an induced-fit scenario for initial events, whereas both scenarios are conceivable for later activation phases as well as for GPCRs activated by diffusible ligands. 4.2.2 Rhodopsin Activation The steps involved in rhodopsin activation are illustrated in Figures 24–26. In the rhodopsin dark state, 11-cis-retinal is tightly bound as an inverse agonist in its binding pocket and covalently fixed by the RSBH+ to Lys296 on TM7. The positively charged RSBH+ is stabilized by a complex counterion comprising negatively charged residues Glu113 on TM3 and Glu181 on extracellular loop 2, 505,506 with the former functioning as the primary counterion (Figure 25A). The β-ionone ring at the other end of the retinal is ensconced in a hydrophobic pocket formed by aromatic side chains, and the conjugated double-bond system linking the two is tightly engaged by a constriction in the retinal binding site which forces a negative 6-s-cis twist of the β-ionone ring about the C6—C7 single bond and a twist about the C11=C12 double bond. 46,420,421 A twist about the C12—C13 single bond results from a steric interaction between a proton at C10 and the methyl group at C20. In addition to the pretwist of the C11=C12 double bond, the proximity of the negatively charged Glu181, which was predicted from modeling, 507 reduces the bond order further to enable selective isomerization around the C11=C12 double bond in the direction shown in Figure 23. Following the gradual release of the potential energy stored in the distorted retinal–protein complex via Batho and the transient BSI, it is not until Lumi that a displacement of the β-ionone ring is observed as a result of the elongation of the retinal. The local perturbations of amino acid side chains in Lumi increase slightly when compared with Batho, but the protein structural changes are still limited to the residues making up the retinal binding site and do not propagate to the cytoplasmic surface. 146 FTIR studies using site-directed infrared labels suggest that the first global movement of the protein backbone is a small rotation of TM5 and TM6 which occurs upon Meta I formation. 479 Solid-state NMR experiments on Meta I and changes in electron density in TM6 on the side facing retinal in the Meta I electron crystallography structure are consistent with a slight motion of TM6 491 which can be described as rotation, but not outward movement. 480 Also consistent with this, another rhodopsin-specific constraint, the TM3/TM5 hydrogen bonding network including Glu122 and Trp126 on TM3 and His211 on TM5, changes upon formation of Meta I as concluded from FTIR data. 508 Straightening of the retinal due to isomerization is thought to move the β-ionone ring toward the region of Met207 to Phe212 on TM5 and thus driving TM rearrangement. 480,509,510 Figure 26 Structural and functional changes in the activation pathway of bovine Rho based on structural and complementary biophysical data discussed in the text and ref (490) and described in Figures 23 and 24. Until formation of Meta I, the RSBH+ remains protonated, but structural changes in the RSB region occur. In the rhodopsin dark state, RSBH+ interacts with the negatively charged Glu113 counterion (Figure 25A) 511−513 from which a hydrogen bonding network extends to Glu181 on extracellular loop 2. 46 On the basis of both UV–vis and Raman spectroscopy it was hypothesized that in Meta I Glu181 transfers a proton to Glu113 and the RSBH+ switches counterions from Glu113 to Glu181. 514 This view was later modified on the basis of FTIR, 505,515 NMR, 516 and molecular dynamics simulations 517 whose data argue for a complex-counterion made up by Glu113 and Glu181, with both residues being deprotonated and giving the retinal binding pocket a net negative charge. 505,516 In the complex-counterion switch model, Glu113 functions as the primary counterion in the photointermediates up to Lumi; in Meta I, the conformational changes in retinal lead to a shift of the counterion from Glu113 to Glu181. 505,515,517 With the deprotonation of the RSB, an equilibrium of Meta II states is reached, in which the RSB nitrogen is deprotonated prior to the occurrence of TM movements. 488,490 As a result of retinal isomerization, the RSBH+ reorients (Figure 23), and its high pK a (determined experimentally to be above 16 in the dark state 518 and suggested to have contributions from the negatively charged Glu181 505,506 ) drops so that the proton dissociates for the formation of the first Meta II state, Meta IIa. Concomitantly, Glu113 becomes protonated, and a direct internal proton transfer from the RSBH+ is likely to be the source of this proton. 519 According to FTIR studies, 490 RSBH+ deprotonation upon transition to Meta IIa leads to a rearrangement of the TM1/2/7 network as seen by changes of infrared bands assigned to Asp83 on TM2. In this water-mediated hydrogen-bonding network, Asp83 (on TM2) links Asn55 (on TM1) with the NPxxY(x)5,6F motif (TM7), and extends to Trp265 (on TM6) adjacent to retinal’s β-ionone ring. Activating conformational changes in the TM3/TM5 network occur in the Meta IIa → Meta IIb transition, as seen by changes of infrared bands assigned to Glu122. 490 This hydrogen bonding network links Glu122 and Trp126 (both on TM3) with His211 (on TM5) which is in contact with the β-ionone ring of retinal. Retinal movement toward TM5 exerts its full effect upon transition to Meta IIb, reflected in a weakening of the hydrogen bond to Glu122. 508,520 In the Meta IIa → Meta IIb transition, changes in infrared amide I marker bands indicate structural changes in the protein backbone. 490 According to Meta II structures, these structural alterations include an elongation of TM5 by 1.5 to 2.5 helix turns depending on the dark state reference structure (PDB ID: 1GZM/3C9L or 1U19, respectively) and a rotational tilt of the kinked TM6 where the TM rotation results in the 6–7 Å outward movement of the cytoplasmic end of TM6 (Figure 23). 426,429,433,435 Time-resolved EPR studies with a spin label sensor at position 227 on TM5, designed to detect TM6 movement, probed the Meta IIa → Meta IIb transition for this event, although the sensor might have detected movements of both TM5 and TM6. 488 TM6 rotation is facilitated by the breakage of the (D/E)RY ionic lock between TM3 and TM6 consisting of Glu134 and Arg135 (on TM3) and Glu247 and Thr251 (on TM6). The cytoplasmic end of TM5 contains two residues, Tyr223 and Lys231, which function as microswitches. 521 These residues face the lipid environment in the dark state, but stabilize the Meta IIb state when the Arg135-Glu247 ionic lock has been disrupted and TM5 and TM6 rearrangements have occurred (Figure 25C,D). By swiveling inward, Tyr223 can hydrogen bond to Arg135 of the ionic lock, thereby linking TM3 and TM5. Glu247 (on TM6) is freed in Meta IIb from Arg135 and can form a salt bridge to Lys231 on TM5, thus stabilizing TM6 in the outward position. Also in the transition to Meta II when TM6 moves, the electrostatic interaction between Tyr306 and Phe313 becomes disrupted allowing Tyr306 to swivel into the vacated space below Arg135 where it becomes part of an extended water-mediated hydrogen bonding network reaching from the retinal binding site via Ser298, Asp83, Asn302, Met257, Tyr306, and Tyr223 to Arg135. 435 Studies with mutants of the NPxxY(x)5,6F motif suggested dual roles for the NP and the Y(x)5,6F submotifs. 522 Whereas the first has a structural role related to the hydrogen bonding network, the latter is involved in the interaction with G protein. The crystal structure revealed how Tyr306 stabilizes Arg135 and thus the cytoplasmic cleft for binding of the Gtα C-terminus. In the structure of the Meta II·GαCT2 peptide complex the backbone carbonyls of Cys347 and Lys345 (on GαCT2) form hydrogen bonds to Arg135 and Gln312, respectively. The presence of the GαCT2 peptide in the cytoplasmic G protein binding cleft moves the cytoplasmic end of TM7 slightly toward the center, leading to a somewhat narrower binding cleft. The proposed water-mediated hydrogen bonding network provides an answer regarding how the retinal binding site and the G protein binding site 30 Å apart are connected. 426,435,464,465 4.2.3 Retinal Channeling in Rhodopsin The structure of bovine Ops* revealed that the retinal binding pocket has two openings toward the lipid bilayer, one opening between TM1 and 7, the other between TM5 and 6, which form the two halves of the channel leading to the retinal binding pocket (Figure 27). Using molecular docking of retinal, a 3–12 Å wide continuous channel through opsin with the retinal binding pocket as the central part was found. 523,524 The openings to these channels are lined with aromatic residues while the central part is more polar. A major constriction of the channel is around Lys296 which enforces a 90° elbow-like kink in the channel. Passage of that restriction would be easier for the kinked 11-cis-retinal, whereas the more elongated and rigid all-trans-retinal would require global conformational changes. A study with rhodopsins containing mutations throughout the channel failed to correlate specific opening(s) with retinal entry and exit. 525 However, the study suggested that the ease of retinal passage through constrictions in the channel is not rate-limiting for rhodopsin reconstitution or Meta II decay, but for RSB formation and hydrolysis, respectively. Upon rhodopsin activation, when the helix bundle opens up, bulk solvent molecules obtain access to the RSB, 526−528 most likely from the cytoplasmic side of rhodopsin for RSB hydrolysis and retinal release. 526 This hypothetical solvent channel appears to be quite narrow because only small nucleophiles such as water and hydroxylamine have access to the RSB for retinal hydrolysis. 526,529 Figure 27 Retinal channel in the Ops*/Meta II conformation. (A) Meta II structure (PDB ID: 3PXO) with the putative retinal channel indicated, rotated to face opening one (red arrow and half channel indicated in red mesh) which is located between TM1 and TM7, and (B) rotated to face opening two (green arrow and half channel indicated in green mesh) which is located between TM5 and TM6. Channels were determined using MOLE 577 on the Ops* structure (PDB ID: 3CAP). The Meta II structure was used for the figure so that the retinal could be shown as it is absent in the Ops* structure. The size of the opening of the retinal channel varies in different Ops*, Meta II, and Meta II-like structures due to variations in side chain rotamers. In the rhodopsin dark state with its compact helix bundle, and presumably in the conformation of inactive opsin, which is similar to dark state rhodopsin, 530 no opening is observed and the 11-cis-retinal appears to reside in a hermetically sealed retinal binding pocket (Figure 28A). 46,420,421 Opsin crystals in the Ops* conformation with an open channel therefore allowed reconstitution of Meta II in soaking experiments with all-trans-retinal. 426 All-trans-retinal can bind tightly in its binding pocket in Meta II with only slight adjustment of the surrounding amino acid side chains. The binding pocket, however, is flexible enough to allow retinal rotation along its long axis upon rhodopsin activation as concluded from the Meta II crystal structures. 426,433 A limited data set of intramolecular distances obtained by solid-state NMR experiments on Meta II is mostly consistent with the crystal structure. A different degree of retinal rotation in the NMR experiment, however, cannot be ruled out, but could be explained by the equilibrium of Meta II substates. 174,426,509,510,531 Figure 28 Retinal binding site of bovine rhodopsin. (A) Crystal structure of inactive dark state (PDB ID: 1U19) where 11-cis-retinal is tightly bound deep in the protein with no openings of the retinal binding site toward the lipidic environment. (B) In the active Ops* (or Meta II) conformation the retinal channel allows all-trans-retinal access and egress, and some detergents like β-d-octylglucoside, mimicking the all-trans-retinal chromophore, to enter the retinal binding site. Shown is an overlay of the two ligands in the retinal binding pocket as observed in the crystal structures of Meta II (all-trans-retinal depicted in yellow; PDB ID: 3PXO) and Ops* in complex with β-d-octylglucoside (depicted in green/red; two rotamers of Lys296 are shown in red; PDB ID: 4J4Q). Note that the ring moieties of chromophore and detergent are oriented in opposite directions. Whereas all-trans-retinal is covalently linked by the RSB to Lys296, β-d-octylglucoside is fixed in the ligand binding site by hydrogen bonding of its hydroxyl groups to the opsin environment. After uptake of 11-cis- or 9-cis-retinal by opsin (likely by the Ops* conformation when openings to the retinal channel are present) and RSB formation, a conformational change yields inactive rhodopsin or isorhodopsin, respectively, both of which generate the same photoproducts after photon absorption. 46,420,421,532 For the formation of artifical rhodopsin pigments, the retinal channel in opsin is flexible enough to take up a wide variety of bulkier retinal analogues, e.g., featuring larger or additional alkyl groups or alkyl rings which prevent isomerization around the C11=C12 double bond. 533 Recently, crystallographic evidence was provided that the detergent, β-d-octylglucoside, can bind within rhodopsin’s retinal binding pocket thus stabilizing the Ops* conformation (Figure 28B). 432 A study on rhodopsin reconstitution from bovine opsin and 11-cis-retinal in the presence of various glucose- or maltose-containing detergents suggested that even some maltoside detergents can enter the retinal channel, while the affinity of various detergents for opsin was dependent on the alkyl chain length. 432 The varied hydrogen-bonding possibilities of the glucose hydroxyl groups to opsin within the retinal binding pocket is reminiscent of the proposed dynamic binding of odorants within olfactory receptors. 534 With its lateral ligand entry and its active GPCR conformation, Ops* was suggested to be ideal for homology modeling of olfactory receptors binding olfactant agonists. 432 4.3 Mechanistic Variations in Other Rhodopsins 4.3.1 Color Pigments Rod and cone cells are responsible for scotopic and photopic vision, i.e., vision under low light and daylight conditions, respectively. Rods are characterized by high sensitivity, slow response, slow dark adaptation, and a single type of rhodopsin, whereas cones have low sensitivity, fast response, fast dark adaptation, and several types of cone rhodopsins. 3,535 Some of the features of cone cells can be attributed to the cone visual pigments, which absorb at different wavelengths [red (λmax = 560 nm), green (λmax = 530 nm), and blue (λmax = 420 nm) rhodopsins in humans] necessary to discriminate colors for color vision. The vertebrate cone opsins and rod rhodopsins (λmax = 500 nm) form a single family of homologous proteins, 26 where rod rhodopsins have evolved from cone visual pigments, being closer to the short-wavelength cone opsins. 536,537 The extinction coefficient and quantum yield and thus photosensitivity of bovine Rho and chicken green pigment are comparable. 538 Cone rhodopsins are also thought to undergo a photoactivation process similar to rod rhodopsin, with some variation for the long wavelength pigments. 539,540 A difference, however, is a shorter lifetime of the Meta II state, with parallel formation of Meta III (a late nonproductive photointermediate involved in the decay of the photoactivated state to the apoprotein opsin and retinal) from Meta I (which is in equilibrium with Meta II, Figure 22). 538,541,542 Additionally, regeneration of cone opsins with 11-cis-retinal is faster than that of rod rhodopsins. 538,543 The faster kinetics of cone opsins (by 1–2 orders of magnitude) of the active state decay and rhodopsin regeneration are optimal and necessary for the high light levels present during the daytime. Site-directed mutagenesis studies identified amino acids at position 122 and 189 (Glu122 and Ile189 in bovine Rho, replaced by Gln/Ile and Pro in cone opsins) to be responsible for this functional difference. 544,545 In bovine Rho, Glu122 is a part of a hydrogen bonding network with His211 (TM3/5 network). 546 Glu122 and Ile189, present in rhodopsin but not in cone pigments, also potentiate a more efficient G protein activation compared with cone pigments. 542 The lower Gt activation capacity of cone pigments and faster Meta II decay contribute to the lower photosensitivity of cones compared with rods. Formation and decay of Meta II are related to TM6 movements as outlined above, and EPR experiments could potentially give insights into structural dynamics and conformational changes of color pigments. Unfortunately, because of the difficulties in the sample isolation and preparation of cone pigments, structural studies on these pigments have lagged significantly behind those of bovine Rho. From resonance Raman spectroscopy it is known that the chromophore structure is similar between human green and red sensitive pigments, with both being similar to that of rhodopsin. 547 From FTIR spectroscopy it was concluded that the protein structure of monkey green and red pigments is clearly different from that of rhodopsin, and that hydrogen-bonding networks differ between green and red pigments. 145 It should be noted that in human red and green color vision pigments Glu181 (in bovine Rho) is replaced by a His residue that functions as a chloride binding site, 548,549 suggesting that participation of an anion is a prerequisite for seeing red light. Future structural and modeling studies will be needed to further elucidate the molecular mechanism(s) of color tuning and structural dynamics in cone pigments. 550 4.3.2 Bistable Rhodopsins and Photoisomerases The rhodopsins we have discussed so far are stable in the dark, but once retinal isomerization has occurred and metarhodopsin states are reached, the eventual decay into opsin and all-trans-retinal takes place. These rhodopsins are also called monostable rhodopsins, and their opsins must be regenerated with new 11-cis-retinal produced in the adjacent retinal pigment epithelium cells. 551 Vertebrates and invertebrates possess in addition to these monostable rhodopsins, a second type of rhodopsins, the bistable rhodopsins, 552 which mostly couple to the G protein, Gq, to initiate phosphoinositol signaling cascades. While this Gq signaling is typical for the majority of bistable rhodopsins including those of squid and octopus, other bistable rhodopsins have been shown to couple to other G proteins such as Go. 3,552 A characteristic of bistable rhodopsins is that the dark state and the active states are both thermally stable; i.e., RSB hydrolysis does not occur. 553 Furthermore, a secondary absorption of a photon is utilized to photoregenerate the dark state. 554 These opsins lack the conserved RSBH+ stabilizing counterion found in monostable rhodopsins (Glu113 on TM3 in bovine Rho). Instead, in squid rhodopsin the RSBH+ forms a hydrogen bond to Asn87 or Tyr111 side chains (the equivalent positions in bovine Rho are Gly89 on TM2 and Glu113 on TM3). 422 In other bistable opsins, position 113 is occupied by neutral amino acid residues such as Tyr, Phe, or Met, which can explain why in contrast to bovine Rho, RSBH+ deprotonation of the active state is not required. 555 The conserved Glu181 (bovine Rho numbering) may serve as the sole negatively charged residue near the RSBH+, which is, however, not close enough for direct interaction with the RSBH+. 422 Molecular evolutionary analysis implies that the counterion has been switched from Glu181 to Glu113 during the evolution of vertebrate opsins and that Glu181 serves as a counterion in bistable pigments. 3,552,556,557 Another distinctive feature of bistable pigments is that their opsins can bind all-trans-retinal to form the pigment. 3,552 Rhodopsin from the Japanese flying squid, T. pacificus is the only invertebrate opsin (and incidentally the only other GPCR purified from native source apart from bovine rhodopsin) to have its structure determined. This structural information has been instrumental in understanding its chromophore–protein interactions in the dark and bathorhodopsin states as well as in the artificial isorhodopsin pigment (containing 9-cis-retinal). 422,423,558 The crystal structures of dark state bovine Rho and squid rhodopsin exhibit similar features including the presence of the disulfide bridge at the extracellular side of the retinal pocket, a hydrophobic aromatic cage surrounding the retinal and the presence of the (D/E)RY (ionic lock) and NPxxY(x)5,6F motifs. 422,423 As in bovine Rho, the retinal is attached through a RSBH+ linkage to the conserved Lys residue on TM7, but retinal itself takes a more relaxed configuration in squid rhodopsin as opposed to the distortions observed in bovine Rho. 422 The structure for squid bathorhodopsin indicates that just as in the bovine Rho early intermediate states, there is little structural change except for a change in the twist of the retinal. 558 It has been postulated from protein structure and sequence analysis that the extended TM5 and TM6 observed in the squid rhodopsin structure may explain the selectivity of coupling to Gq proteins. 422,559 Squid rhodopsin contains an additional C-terminal domain that might be involved in G protein binding, but it was not structurally characterized as it was necessary to proteolytically remove this domain comprising the last 90 amino acids for crystallization. 422,423 Another reason for the functional difference may be the extent of TM6 movement upon photoactivation of bistable rhodopsins as proposed from site-directed fluorescence labeling measurements. A comparison of bovine Rho and parapinopsin, a bistable nonvisual Gt-coupled rhodopsin, revealed much smaller light-induced TM5 and TM6 movement for parapinopsin which correlates with its reduced capability to activate G protein. 560 Of further interest among Gq-coupled bistable rhodopsins is melanopsin, which has been identified in various vertebrates. 552,561 In mammals, melanopsin is localized in intrinsically photosensitive retinal ganglion cells and is involved in nonvisual functions, including photoentrainment of the circadian clock, pupillary light reflex, and sleep. 4 Another, more divergent grouping of opsins are the photoisomerases, which function to bind all-trans retinoids and photoisomerize them to their 11-cis-forms. 410 Structural information remains elusive for these opsins. The best characterized of these photoisomerases are retinochrome from molluscan species and the mammalian retinal G-protein-coupled receptor (RGR). 97,562,563 In mollusks, retinochrome functions to provide the 11-cis-retinal to newly synthesized apoprotein opsin, whereas in mammals, RGR appears to play more of a regulatory role in the mobilization of all-trans-retinyl esters into the retinoid cycle. 562,564 These photoisomerases lack the NPxxY(x)5,6Y motif and thus may be deficient in G protein coupling. 3 A close relative is peropsin from mammalian retinal pigment epithelium (RPE) cells. Peropsin shows photoisomerase activity, but contains the (D/E)RY and NPxxY(x)5,6Y motifs and therefore may couple to G protein and be involved in activation of signaling cascades. 565 5 Conclusions and Perspective In the past, the impact of research on both microbial and animal rhodopsins propagated far beyond the boundaries of the retinal-binding protein field. It will suffice to give just a few of the most striking examples. Structural work on BR and Rho has greatly contributed to our understanding of the structural principles of membrane proteins in general. The detailed understanding of the proton transport mechanism by BR has been extremely useful to the bioenergetics community, who extended these principles to important systems such as cytochrome oxidases and ATPases. The great structural and mechanistic advances in the understanding of visual signal transduction significantly enriched the GPCR field, and for many years bovine Rho served (and continues to serve) as a model GPCR. More recently, the discovery of proteorhodopsins gave a strong push to the field of metagenomics, while the discovery of channelrhodopsins gave birth to optogenetics. Finally, rhodopsins have been serving as testing grounds for many cutting-edge biophysical techniques, aiding, for example, in the development of time-resolved and low-temperature FTIR spectroscopy, ultrafast spectroscopy, advanced Raman techniques, new methods for 2-D and 3-D crystallization of membrane proteins, protein solid-state NMR, and high-field EPR, including site-directed spin-labeling techniques. But what are the next exciting steps in rhodopsin research? Expanding on these recent trends, we can predict many more interesting developments without being too speculative. Judging from the large number of new rhodopsin variants unearthed by genomic and metagenomic sequencing, new interesting functions of retinal proteins will continue to be discovered. This applies to both microbial and animal rhodopsins, and will lead to new breakthroughs in understanding microbial, invertebrate, and vertebrate physiology and evolution. These new functional variants of rhodopsins may also find use in optogenetics, enriching its arsenal of tools. New advanced techniques of structural biology and biophysics will be applied to these new rhodopsins leading to new insights, capitalizing on such emerging methods as, for example, high-speed AFM, ultrafast time-resolved crystallography, structural mass spectroscopy, and dynamic nuclear polarization NMR. From the point of view of structural biology, the next challenging frontier in the field will be to understand the mechanisms of rhodopsin–protein interactions. While structural methods have enjoyed great success in determining structures of isolated rhodopsins and their binding partners, structures of inactive and activated protein complexes, especially those of membrane and soluble proteins, remain elusive. This is especially important for visual rhodopsins with their multiple interacting partners, and bears on the entirety of the GPCR field, contributing to a better understanding of GPCR signaling cascade, activation, and regulation.