Rhodopsin is a transmembrane protein found in the rodcells of the retina and is also a G-protein coupled receptor. It belongs to alarger family of light-sensitive proteins called opsins. The primary function ofrhodopsin is to enable vision, particularly in low levels of light, via the conversionof photons into a nerve signal, i.
e. the visual transduction pathway. This isachieved with the assistance of its cofactor – retinal – and a heterotrimericG-protein called transducin. Bovine rhodopsin will be discussed; however, it isstructurally and functionally similar to all animal rhodopsins.
The main structural components of rhodopsin are itsprotein component, opsin, and a covalently-bound cofactor called retinal. Wheninactive, the retinal is in its 11-cis configuration. This is a stereoisomerwhere the 2 functional groups of the 11th and 12th carbonare on the same side of the double bond. The protein component consists of 348amino acids that are arranged in 7 transmembrane ?-helices (TMHs) which arejoined by 3 extracellular protein loops and 3 cytoplasmic protein loops (Palczewski,2006). A disulphide bridge is also present that links one of the extracellularloops to a TMH. Due to the irregular conformation of the TMHs, variousinterhelical interactions such as hydrogen bonding and salt bridges are presentto confer greater stability (Caltabiano et al.
, 2013). The N-terminus islocated on the extracellular side while the C-terminus is located on thecytoplasmic side. In addition to the 7 TMHs, an amphipathic 8thhelix is present in the cytoplasm as a component of the C-terminus (Teller etal.
, 2001). A summary of the overall structure is shown in figure 1:Figure Two: thestructure of 11-cis retinal with its protonated Schiff base linkage. A skeletalstructure is shown with the carbons numbered except for the Schiff base linkagewhich is in blue. (Modified from Berg, Stryer & Tymoczko, 2012) A negatively-chargedglutamate residue (E113) on helix III acts as a counterion and furtherstabilises the linkage.
This residue is involved in a hydrogen-bonding networkwith another glutamate residue (E181), a serine residue and 2 water molecules (Tsutsui& Shichida, 2010) Apart from the Schiff base linkage, other structuralfeatures are present in rhodopsin. Towards the cytoplasmic face of helix III, ashort sequence of amino acid residues consisting of glutamate, arginine andtyrosine (ERY) is present. While rhodopsin is inactive, a salt bridge ispresent that links the glutamate and arginine residues as well as anotherglutamate residue on helix VI (Filipek et al., 2003). This is important in theconformational changes associated with rhodopsin photoactivation as thedissociation of the salt bridge facilitates the binding of transducin (Mahalingamet al.
, 2008). Another important structural feature is the 2 oligosaccharidechains bonded to the 2 asparagine residues near the N-terminus. Theoligosaccharide chains consist of 3 mannose residues and 3 N-acetylglucosamineresidues which are covalently added via N-linked glycosylation in theendoplasmic reticulum and Golgi apparatus. The purpose of this is to ensure thatrhodopsin is correctly folded and so can function properlMT1 y.(Kaushal, Khorana & Ridge, 1994). Rhodopsin typically exists in the disc membrane in theouter segment of rod cells. While this is separate from the plasma membrane, itis still composed of a phospholipid bilayer (Palczewski, 2006). The amino acidsequences of the 7 TMHs indicate that most of the residues that are closer tothe centre of the helix, and hence embedded within hydrophobic phospholipidtails, are hydrophobic residues such as leucine, tyrosine and phenylalanine(Huber et al.
, 2004). Grouping hydrophobic structures is more energeticallyfavourable as it minimises disruption to the cytoplasmic hydrogen bondingnetwork of water, thereby lowering entropic costs. In contrast, residuestowards the cytoplasmic and extracellular faces are embedded within the hydrophilichead groups of phospholipids and therefore tend to be polar. A post-translationmodification also contributes to the amphipathic properties of rhodopsin: helixVIII is anchored to the hydrophobic tails of the phospholipid bilayer by amodification called palmitoylation.
This is a reaction catalysed by proteinacyl transferase where a long-chain fatty acid called palmitate is transferredfrom coenzyme A to the thiol groups of 2 cysteine residues (C322 and C323) toform 2 thioester linkages in an esterification reaction (Filipek et al., 2003).Additionally, the hydrophobic binding site of retinal is embedded within thephospholipid bilayer; it is surrounded by the hydrophobic amino acid residuesof helices V and VI (Zhou, Melcher & Xu, 2012). Because retinal is ahydrophobic molecule, these residues provide greater stability to retinal whileit is bound.
The mechanism of action for rhodopsin is triggered bythe photoisomerisation of retinal from its 11-cis stereoisomer to its all-transstereoisomer. This results in conformational changes in rhodopsin via severalintermediates. The first intermediate is photorhodopsin while the remainingintermediates are denoted by the prefixes: batho, lumi, meta I, meta II.
The protonationof the Schiff base linkage is crucial as it shifts the wavelength of photonsabsorbed from 380nm, corresponding to the UV spectrum, to 440nm – correspondingwith visible light (Berg, Stryer & Tymoczko, 2012). The absorption ofphotons then provides energy for electron transitions from ?-bonding orbitalsto ?-antibonding orbitals in the conjugated system of 11-cis retinal (Zhou,Melcher & Xu, 2012), thus lowering the activation energy for photoisomerisationand thereby facilitating rotation around the C11-C12 double bond. This rotationis largely due to the change in the dihedral angle around the bond (Nakamichi& Okada, 2006). The resulting twisted conformation of all-trans retinalstores two-thirds of the energy absorbed, corresponding to 238 kJmol-1(Okada et al.
, 2001). The energy stored in this strained conformation isreleased as all-trans retinal thermally decomposes to a non-strainedconformation, providing the necessary energy for irreversible protein conformationalchanges that result in the formation of the meta I intermediate (Palczewski,2014). These changes primarily affect the hydrophobic binding site of retinal.
Upon formation of the meta I intermediate, the Schiffbase linkage remains protonated. However, it is thought that its counterionchanges from E113 to E181 due to the conformational changes of the retinalbinding site (Ernst et al., 2014). Because the pKa of the Schiffbase linkage is very high in inactive rhodopsin, the change of counterion isnecessary for the lowering of its pKa to facilitate itsdeprotonation (Lüdeke et al., 2005). This process leads to the protonation of E113 and the formation of themeta II intermediate. Another conformational change in the protein arisesbecause of this; the hydrogen-bonding network between helices I, II and VII isdisrupted and rearranged, resulting in a change of position of retinal in itsbinding site (Zaitseva, Brown & Vogel, 2010). This change in positionfurther rearranges hydrogen-bonding between helices III and V.
Most notably,the uptake of a proton from the solution breaks the ERY salt bridge byprotonation of the glutamate residue, causing a change in the position of helixVI which opens a cytoplasmic binding site for transducin (Ernst et al., 2014).Studies using a synthetic peptide derived from theC-terminus of the ?-subunit of transducin have shown that the binding of the?-subunit to rhodopsin involves interactions with the hydrophobic amino acidresidues of helix VI (Zhou, Melcher & Xu, 2012). It also involves hydrogenbonds between carbonyl groups in the peptide bonds of the ?-subunit and anarginine residue on helix III as well as a glutamine residue on helix VIII(Ernst et al., 2014).
This binding catalyses the exchange of GDP for GTP on the?-subunit. The ?-subunit then dissociates from rhodopsin and its ?- and ?-subunits and activates a downstream signalling cascade, ultimately leading tovision. Following this, the Schiff base linkage is hydrolysed, releasingall-trans retinal (Scheerer et al., 2008). This is subsequently replaced by amolecule of 11-cis retinal to restart the signalling cascade.
In summary, rhodopsin is a transmembrane G-proteincoupled receptor with amphipathic properties that allow it to exist in the discmembrane of rod cells. The photoisomerisation of its cofactor – retinal –trigger a series of conformational changes that ultimately activate transducin,leading to the production of a nerve signal for vision.