Rhodopsin in 7 transmembrane ?-helices (TMHs) which are

Rhodopsin is a transmembrane protein found in the rod
cells of the retina and is also a G-protein coupled receptor. It belongs to a
larger family of light-sensitive proteins called opsins. The primary function of
rhodopsin is to enable vision, particularly in low levels of light, via the conversion
of photons into a nerve signal, i.e. the visual transduction pathway. This is
achieved with the assistance of its cofactor – retinal – and a heterotrimeric
G-protein called transducin. Bovine rhodopsin will be discussed; however, it is
structurally and functionally similar to all animal rhodopsins.

The main structural components of rhodopsin are its
protein component, opsin, and a covalently-bound cofactor called retinal. When
inactive, the retinal is in its 11-cis configuration. This is a stereoisomer
where the 2 functional groups of the 11th and 12th carbon
are on the same side of the double bond. The protein component consists of 348
amino acids that are arranged in 7 transmembrane ?-helices (TMHs) which are
joined by 3 extracellular protein loops and 3 cytoplasmic protein loops (Palczewski,
2006). A disulphide bridge is also present that links one of the extracellular
loops to a TMH. Due to the irregular conformation of the TMHs, various
interhelical interactions such as hydrogen bonding and salt bridges are present
to confer greater stability (Caltabiano et al., 2013). The N-terminus is
located on the extracellular side while the C-terminus is located on the
cytoplasmic side. In addition to the 7 TMHs, an amphipathic 8th
helix is present in the cytoplasm as a component of the C-terminus (Teller et
al., 2001). A summary of the overall structure is shown in figure 1:

Figure Two: the
structure of 11-cis retinal with its protonated Schiff base linkage. A skeletal
structure is shown with the carbons numbered except for the Schiff base linkage
which is in blue. (Modified from Berg, Stryer & Tymoczko, 2012)

 A negatively-charged
glutamate residue (E113) on helix III acts as a counterion and further
stabilises the linkage. This residue is involved in a hydrogen-bonding network
with another glutamate residue (E181), a serine residue and 2 water molecules (Tsutsui
& Shichida, 2010)  

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Apart from the Schiff base linkage, other structural
features are present in rhodopsin. Towards the cytoplasmic face of helix III, a
short sequence of amino acid residues consisting of glutamate, arginine and
tyrosine (ERY) is present. While rhodopsin is inactive, a salt bridge is
present that links the glutamate and arginine residues as well as another
glutamate residue on helix VI (Filipek et al., 2003). This is important in the
conformational changes associated with rhodopsin photoactivation as the
dissociation of the salt bridge facilitates the binding of transducin (Mahalingam
et al., 2008). Another important structural feature is the 2 oligosaccharide
chains bonded to the 2 asparagine residues near the N-terminus. The
oligosaccharide chains consist of 3 mannose residues and 3 N-acetylglucosamine
residues which are covalently added via N-linked glycosylation in the
endoplasmic reticulum and Golgi apparatus. The purpose of this is to ensure that
rhodopsin is correctly folded and so can function properlMT1 y.
(Kaushal, Khorana & Ridge, 1994).  

Rhodopsin typically exists in the disc membrane in the
outer segment of rod cells. While this is separate from the plasma membrane, it
is still composed of a phospholipid bilayer (Palczewski, 2006). The amino acid
sequences of the 7 TMHs indicate that most of the residues that are closer to
the centre of the helix, and hence embedded within hydrophobic phospholipid
tails, are hydrophobic residues such as leucine, tyrosine and phenylalanine
(Huber et al., 2004). Grouping hydrophobic structures is more energetically
favourable as it minimises disruption to the cytoplasmic hydrogen bonding
network of water, thereby lowering entropic costs. In contrast, residues
towards the cytoplasmic and extracellular faces are embedded within the hydrophilic
head groups of phospholipids and therefore tend to be polar. A post-translation
modification also contributes to the amphipathic properties of rhodopsin: helix
VIII is anchored to the hydrophobic tails of the phospholipid bilayer by a
modification called palmitoylation. This is a reaction catalysed by protein
acyl transferase where a long-chain fatty acid called palmitate is transferred
from coenzyme A to the thiol groups of 2 cysteine residues (C322 and C323) to
form 2 thioester linkages in an esterification reaction (Filipek et al., 2003).
Additionally, the hydrophobic binding site of retinal is embedded within the
phospholipid bilayer; it is surrounded by the hydrophobic amino acid residues
of helices V and VI (Zhou, Melcher & Xu, 2012). Because retinal is a
hydrophobic molecule, these residues provide greater stability to retinal while
it is bound.

The mechanism of action for rhodopsin is triggered by
the photoisomerisation of retinal from its 11-cis stereoisomer to its all-trans
stereoisomer. This results in conformational changes in rhodopsin via several
intermediates. The first intermediate is photorhodopsin while the remaining
intermediates are denoted by the prefixes: batho, lumi, meta I, meta II. The protonation
of the Schiff base linkage is crucial as it shifts the wavelength of photons
absorbed from 380nm, corresponding to the UV spectrum, to 440nm – corresponding
with visible light (Berg, Stryer & Tymoczko, 2012). The absorption of
photons then provides energy for electron transitions from ?-bonding orbitals
to ?-antibonding orbitals in the conjugated system of 11-cis retinal (Zhou,
Melcher & Xu, 2012), thus lowering the activation energy for photoisomerisation
and thereby facilitating rotation around the C11-C12 double bond. This rotation
is largely due to the change in the dihedral angle around the bond (Nakamichi
& Okada, 2006). The resulting twisted conformation of all-trans retinal
stores two-thirds of the energy absorbed, corresponding to 238 kJmol-1
(Okada et al., 2001). The energy stored in this strained conformation is
released as all-trans retinal thermally decomposes to a non-strained
conformation, providing the necessary energy for irreversible protein conformational
changes 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 Schiff
base linkage remains protonated. However, it is thought that its counterion
changes from E113 to E181 due to the conformational changes of the retinal
binding site (Ernst et al., 2014). Because the pKa of the Schiff
base linkage is very high in inactive rhodopsin, the change of counterion is
necessary for the lowering of its pKa to facilitate its
deprotonation (Lüdeke et al., 2005). 
This process leads to the protonation of E113 and the formation of the
meta II intermediate. Another conformational change in the protein arises
because of this; the hydrogen-bonding network between helices I, II and VII is
disrupted and rearranged, resulting in a change of position of retinal in its
binding site (Zaitseva, Brown & Vogel, 2010). This change in position
further rearranges hydrogen-bonding between helices III and V. Most notably,
the uptake of a proton from the solution breaks the ERY salt bridge by
protonation of the glutamate residue, causing a change in the position of helix
VI which opens a cytoplasmic binding site for transducin (Ernst et al., 2014).

Studies using a synthetic peptide derived from the
C-terminus of the ?-subunit of transducin have shown that the binding of the
?-subunit to rhodopsin involves interactions with the hydrophobic amino acid
residues of helix VI (Zhou, Melcher & Xu, 2012). It also involves hydrogen
bonds between carbonyl groups in the peptide bonds of the ?-subunit and an
arginine 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 to
vision. Following this, the Schiff base linkage is hydrolysed, releasing
all-trans retinal (Scheerer et al., 2008). This is subsequently replaced by a
molecule of 11-cis retinal to restart the signalling cascade.

In summary, rhodopsin is a transmembrane G-protein
coupled receptor with amphipathic properties that allow it to exist in the disc
membrane 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.