The irreducible process of phototransduction, 11 cis retinal synthesis, and the visual cycle, essential for vertebrate vision
William Bialek: More Perfect Than We Imagined – March 23, 2013
Excerpt: photoreceptor cells that carpet the retinal tissue of the eye and respond to light, are not just good or great or phabulous at their job. They are not merely exceptionally impressive by the standards of biology, with whatever slop and wiggle room the animate category implies. Photoreceptors operate at the outermost boundary allowed by the laws of physics, which means they are as good as they can be, period. Each one is designed to detect and respond to single photons of light — the smallest possible packages in which light comes wrapped. “Light is quantized, and you can’t count half a photon,” said William Bialek, a professor of physics and integrative genomics at Princeton University. “This is as far as it goes.” … In each instance, biophysicists have calculated, the system couldn’t get faster, more sensitive or more efficient without first relocating to an alternate universe with alternate physical constants. 9
From the book: Evolution of Visual and Non-visual Pigments, page 106
Opsin—the protein that underlies all animal vision., has become a favorite research target, not only of vision scientists but of many researchers interested in the evolution of protein structure, function, and specialization. This level of focus has made the opsins canonical G-protein-coupled receptors (GPCRs) and arguably the most investigated protein group for its evolutionary radiations and diverse functional specializations. Still, opsin’s early evolution REMAINS PUZZLING, and there are many questions throughout its evolutionary history for which we have partial, but tantalizingly incomplete, answers. Obviously, the invertebrates, with their astonishing diversity and with evolutionary hints of the most ancient animals in their genomes, functions, and even body plans, offer the best hope of answering many of these fundamental questions.
Rhodopsins and Cone opsins have two interdependent agents, namely 11 cis retinal chromophores, and opsins, to which they are attached. By absorbing a photon, 11 cis retinal isomerizes to trans retinal conformation, and that triggers a conformational change in opsins, which trigger the signal transduction cascade, which in the end, provokes the electrical signal, transmitted to the brain for processing.
11-cis-Retinal is a unique molecule with a chemical design that allows optimal interaction with the opsin apoprotein in its binding pocket, and this is essential for the formation of the light-activated conformation of the receptor. 2
There are many things that are functionally important, and must be JUST RIGHT, in order for these molecular mechanisms to work.
The fact that rhodopsin has been intensely studied, provides a WEALTH of information on a molecular level, which permits to make INFORMED CONCLUSIONS of its origins.
Now OBSERVE how many things must be JUST RIGHT and ESSENTIAL ( following is straightforward from the relevant scientific literature ) :
Rhodopsin Structure and Activation
Rhodopsin consists of an apoprotein opsin and an inverse agonist ( that’s like a mechanism which keeps a switch off ), the 11-cis-retinal chromophore, which is covalently bound through a Schiff base linkage to the side chain of Lys296 of opsin protein.
The binding of the chromophore to the opsin is essential to trigger the conformational change. That means, there had to be
– a Schiff base linkage
– a Lys296 residue where chromophore retinal covalently binds
– the side chain of the residue
– an essential amino acid residue called “counter ion” key factor appears to be the protonation state of the Schiff-base counterion
– a pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of rhodopsin
– A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. 2
Residues important for stabilizing the tertiary structure
– (e.g. disulphide bridge (S-S),
– amino-terminal (N) glycosylation sites)
– activation/deactivation of photopigments (e.g. carboxyl-terminal (C) phosphorylation sites)
– membrane anchorage (e.g. palmitoylation sites)
For visible light absorption, all opsins contain an essential amino acid residue called “counter ion”, in addition to a retinal-binding site, Lys296 (in the bovine rhodopsin numbering system), where chromophore retinal covalently binds through a protonated Schiff base linkage . The proton on the Schiff base is necessary for visible light absorption, but energetically unstable within the opsin molecule. In opsin pigments, a negatively charged amino acid residue, counterion, stabilizes the protonated Schiff base, and is an essential amino acid residue for opsin pigments to absorb visible light.
Various types of opsin-based pigments with absorption maxima in the visible light region possess a “protonated” Schiff base linkage. In the protein moiety, the positive charge on the protonated Schiff base is unstable, and therefore a counterion, a negatively charged amino acid residue is needed to stabilize the positive charge. In vertebrate visual pigment, glutamic acid at position 113 serves as the counterion 11
Furthermore: movement of the cytoplasmic end of the sixth transmembrane helix is essential for pigment activation.
From the above information, it is clear that there is an evidently FINE- TUNED protein-protein interaction, that is, the 11 cis retinal chromophore physical constitution, and the opsin physical constitution, MUST BE JUST RIGHT from the beginning, and be able to interact PRECISELY to trigger the signal transduction chain.
Let’s suppose, opsin is able to interact with TRANSDUCIN. So what ?? If the signal transduction pathway is not fully setup, and able to go all the way through – no signal – no vision. So having such a precise protein-protein arrangement will make only sense, if down down there, after many complex molecular interactions, a visual image is generated in the brain. After two amplification steps, the goal is achieved, and a signal is sent to the brain. To get that signal, is a REMARKABLE SIGNAL AMPLIFICATION mechanism:
A single photoactivated rhodopsin catalyzes the activation of 500 transducin molecules. Each transducing can stimulate one cGMP phosphodiesterase molecule and each cGMP phosphodiesterase molecule can break down 1000 molecules of cGMP per second. Therefore, a single activated rhodopsin can cause the hydrolysis of more than 100.000 molecules of cGMP per second.
Following enzymes, molecules, and proteins are ESSENTIAL in the signal transduction pathway:
Rhodopsin Rhodopsin is an essential G-protein coupled receptor in phototransduction.
Retinal Schiff base cofactor All-trans-retinal is also an essential component of type I, or microbial, opsins such as bacteriorhodopsin, channelrhodopsin, and halorhodopsin.
Transducin Their function is to mediate the signal transduction from the photoreceptor proteins, the opsins, to the effector proteins, the phosphodiesterases 6
Guanosine diphosphate ( GDP ) Transducin is tightly bound to a small organic molecule called Guanosine diphosphate ( GDP )
Guanosine triphosphate GTP when it binds to rhodopsin the GDP dissociates itself from transducin and a molecule called GTP, which is closely related to, but critically different from, GDP, binds to transducin.
G-nucleotide exchange factor (GEF) The exchange of GDP for GTP is done by a G-nucleotide exchange factor (GEF) 7
Cyclic guanosine monophosphate (cGMP)
phosphodiesterase (PDE) is necessary to transform cGMP to GMP. This closes the cGMP gated ion channel due to the decreasing amounts of cGMP in the cytoplasm 6
cGMP-gated channel of rod photoreceptors
Cyclic nucleotide-gated Na+ ion channels
Once the signal goes through, a system is required to stop the signal that is generated and restore the opsin to its original state. For that task, other essential proteins are needed to restore the initial state of rhodopsin:
The biosynthesis of 11 Cis retinal, essential in the first step of vertebrate vision, is also REMARKABLE.
There is an INTRIGUING EVOLUTIONARY CONSERVATION of the key components involved in chromophore production and recycling, these genes also have adapted to the specific requirements of both insect and vertebrate vision. Visual GPCR signaling is unique with respect to its dependence on a diet-derived chromophore (retinal or 2-dehydro-retinal in vertebrates; retinal and 3-hydroxy-retinal in insects). The chromophore is naturally generated by oxidative cleavage of carotenoids (C40) to retinoids.(C20). Then the retinoid cleavage product must be metabolically converted to the respective 11-cis-retinal derivative in either the same carotenoid cleavage reaction or a separate reaction. 3
All animals endowed with the ability to detect light through visual pigments need pathways in which dietary precursors for chromophore, such as carotenoids and retinoids, are first absorbed in the gut, and then transported, metabolized and stored within the body to establish and sustain vision.
Two fundamental processes in chromophore metabolism defied molecular analysis for a long time: the conversion of the parent C40 carotenoid precursor into C20 retinoids and the all-trans to 11-cis isomerization and cleavage involved in continuous chromophore renewal. Following proteins are essential in the pathway to synthesize 11 cis retinals :
retinal pigment epithelial (RPE) The retinal pigment epithelium (RPE), a single layer of cuboidal cells lying betweenBruch’s membrane and the photoreceptors, is an essential component of the visual system.
Lecithin-retinol acyltransferase Is Essential for Accumulation of All-trans-Retinyl Esters in the Eye and in the Liver 4
Retinyl ester hydrolase
Isomerohydrolase It performs the essential enzymatic isomerization step in the synthesis of 11-cis retinal. 5
RPE retinal G protein-coupled receptor (RGR)
The absorption of light by rhodopsin results in the isomerization of the 11- cis -retinal chromophore to all- trans forming the enzymatically active intermediate, metarhodopsin II, which commences the visual transduction process.
Continuous vision depends on recycling of the photoproduct all-trans-retinal back to visual chromophore 11-cis-retinal. This process is enabled by the visual (retinoid) cycle, a series of biochemical reactions in photoreceptor, adjacent RPE and Müller cells.
Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an UNSOLVED MYSTERY.
Restoration of light sensitivity requires chemical reisomerization of trans-retinal via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE).
When a photon of light is absorbed, 11-cis retinal is transformed to all-trans retinal, and it moves to the exit site of rhodopsin. It will not leave the opsin protein until another fresh chromophore comes to replace it, except for in the ABCR pathway. Whilst still bound to the opsin, all-trans retinal is transformed into all-trans retinol by all-trans Retinol Dehydrogenase. It then proceeds to the cell membrane of the rod, where it is chaperoned to the Retinal Pigment Epithelium (RPE) by Interphotoreceptor Retinoid Binding Protein (IRBP). It then enters the RPE cells, and is transferred to the Cellular Retinol Binding Protein (CRBP) chaperone. 8
The visual cycle fulfills an essential task of maintaining visual function and needs therefore to be adapted to different visual needs such as vision in darkness or lightness. For this, functional aspects come into play: the storage of retinal and the adaption of the reaction speed. Basically vision at low light intensities requires a lower turn-over rate of the visual cycle whereas during light the turn-over rate is much higher. In the transition from darkness to light suddenly, large amount of 11-cis retinal is required. This comes not directly from the visual cycle but from several retinal pools of retinal binding proteins which are connected to each other by the transportation and reaction steps of the visual cycle.
This cycle is present only in vertebrates, as cephalochordates and tunicates do not possess the required enzymes. The isomerization of 11-cis retinal to all-trans retinal in photoreceptors is the first step in vision. For photoreceptors to function in constant light, the all-trans retinal must be converted back to 11-cis retinal via the enzymatic steps of the visual cycle. Within this cycle, all-trans retinal is reduced to all-trans retinol in photoreceptors and transported to the Retinal pigment epithelium (RPE). In the RPE, all-trans retinol is converted to 11-cis retinol, and in the final enzymatic step, 11-cis retinol is oxidized to 11-cis retinal. The first and last steps of the classical visual cycle are reduction and oxidation reactions, respectively, that utilize retinol dehydrogenase (RDH) enzymes.
To make things even more intriguing, there are at least 4 different pathways for regeneration of 11 Cis retinal. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. In the vertebrate cycle, following proteins are ESSENTIAL :
Rhodopsin (also known as visual purple) is a light-sensitive receptor protein involved in visual phototransduction.
Photoreceptor cells are specialized type of cell found in the retina that is capable of visual phototransduction.
Retinal pigment epithelium (RPE) is the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells
Retinal G-protein-coupled receptor (RGR) is a non-visual opsin expressed in RPE. RGR bound to all-trans-RAL is capable of operating as a photoisomerase that generates 11-cis-RAL in the light-dependent manner
Interphotoreceptor retinoid-binding protein (IRBP), an abundant 140 kDa glycoprotein secreted by photoreceptors . The binding of retinoids by IRBP protects them from oxidation and isomerization.
β-Carotene 15,15′-monooxygenase (BCO) in RPE supplies all-trans-RAL to the visual cycle via central cleavage of β-carotene
Cellular retinaldehyde-binding protein (CRALBP) binds 11-cis-ROL and 11-cis-RAL
Retinoid isomerase RPE65 (or isomerohydrolase) in the RPE. RPE65 is involved in the all-trans to 11-cis isomerization.
Retinoids need to be shuttled between different organelles and protected from isomerization, oxidation, and condensation. Thus, key retinoid-binding proteins are critical for maintaining proper retinoid isomeric and oxidation states. Cellular retinaldehyde–binding protein (CRALBP) in the RPE and Müller cells, and extracellular interphotoreceptor retinoid–binding protein (IRBP) are two major carriers involved. The structure of CRALBP—with its unanticipated isomerase activity—has been elucidated, whereas the structure of IRBP has only been partially characterized. Inactivating mutations in either one of these binding proteins can cause retinal degenerative disease. More.