Rhodopsin Molecules Research Articles

A modified approach to the measurement problem: Objective reduction in the retinal molecule prior to conformational change

A new analysis of the measurement problem reveals the possibility that collapse of the wavefunction may now take place just before photoisomerization of the rhodopsin molecule in the retinal rods. It is known that when a photon is initially absorbed by the retinal molecule which, along with opsin comprises the rhodopsin molecule, an electron in the highest π orbital is immediately excited to a π* orbital. This means that a measurement or transfer of information takes place at the quantum level before the retinal molecule commences the conformational change from cis to trans. This could have profound implications for resolving some of the foundational issues confronting quantum mechanics.

Photons, femtoseconds and dipolar interactions: a molecular picture of the primary events in vision.

The first 200 femtoseconds in the life of a photoexcited rhodopsin molecule are extremely important for the development of visual sensation. Immediately upon excitation, a dramatic change in the charge distribution of the cationic 11-cis-retinal protonated Schiff base chromophore occurs that is quantitated by the change in electronic dipole moment of approximately 15 Debye. The opsin protein tunes the absorption maximum of the pigment to the blue or to the red enabling colour vision by placing dipolar rather than charged residues in the chromophore binding site to differentially stabilize either the ground or the excited state charge distribution. Resonance Raman intensity analysis reveals that the 11-cis-retinal chromophore then distorts violently about the C11 = C12 double bond, reaching torsional angles of approximately 70 degrees in only 30 fs. This rapid torsional distortion is driven by the non-bonded interaction between the 13-methyl group and the 10-hydrogen that is unique to the 11-cis configuration of the chromophore. The excited state depopulates in approximately 50 fs through a rapid and vibrationally coherent transition to the ground electronic state manifold with relaxation to the formally trans photoproduct complete in only 200 fs. This unusually fast and efficient isomerization process establishes a new paradigm for condensed phase photochemical reactions.

Reprint of “Crystal packing analysis of Rhodopsin crystals” [J. Struct. Biol. 158 (2007) 455–462

Oligomerization has been proposed as one of several mechanisms to regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallographic analyses of two new crystal forms of rhodopsin reveal an interaction surface which may be involved in the formation of functional dimers or oligomers. New crystallization conditions lead to the formation of two crystal forms with similar rhodopsin–rhodopsin interactions, but changes in the crystal lattice are induced by the addition of different surfactant additives. However, the intermolecular interactions between rhodopsin molecules in these crystal structures may reflect the contacts necessary for the maintenance of dimers or oligomers in rod outer segment membranes. Similar contacts may assist in the formation of dimers or oligomers in other GPCRs as well. These new dimers are compared with other models proposed by crystallography or EM and AFM studies. The inter-monomer surface contacts are different for each model, but several of these models coincide in implicating helix I, II, and H-8 as contributors to the main contact surface stabilizing the dimers.

G Protein-Coupled Receptors Self-Assemble in Dynamics Simulations of Model Bilayers

Many integral membrane proteins assemble to form oligomeric structures in biological membranes. In particular, seven-transmembrane helical G protein-coupled receptors (GPCRs) appear to self-assemble constitutively in membranes, but the mechanism and physiological role of this assembly are unknown. We developed and employed coarse-grain molecular dynamics (CGMD) models to investigate the molecular basis of how the physicochemical properties of the phospholipid bilayer membrane affect self-assembly of visual rhodopsin, a prototypical GPCR. The CGMD method is a mesoscopic simulation technique in which groups of atoms are mapped to particles on the basis of a four-to-one rule. This systematic reduction of the degrees of freedom allows for computationally efficient calculation of the structure and dynamics of molecular assemblies for larger time and length scales than accessible to atomistic models, providing here an unprecedented view of spontaneous protein assembly in biomembranes. Systems with up to 16 rhodopsin molecules at a protein-to-lipid ratio of 1:100 were simulated for time scales of up to 8 micros. The results obtained for four different phospholipid environments showed that localized adaptation of the membrane bilayer to the presence of receptors is reproducibly most pronounced near transmembrane helices 2, 4, and 7. This local membrane deformation appears to be a key factor defining the rate, extent, and orientational preference of protein-protein association. The implications of our findings are discussed within a framework of a generalized mechanism of membrane protein self-assembly.

Coherent signal amplification in rhodopsin media

A quantum optics model that explains the sensitivity of the dark-adapted rhodopsin to a single photon is presented. We describe the absorption of photons by rhodopsin molecules using the three-level A-type model for its retinal chromofore, with 3 states being the all-trans, 11-cis and the photoexcited S 1 state. The abinitio calculations have been performed to estimate the parameters of the model.

Structure and function of the visual arrestin oligomer

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.

Crystal packing analysis of Rhodopsin crystals

Oligomerization has been proposed as one of several mechanisms to regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallographic analyses of two new crystal forms of rhodopsin reveal an interaction surface which may be involved in the formation of functional dimers or oligomers. New crystallization conditions lead to the formation of two crystal forms with similar rhodopsin–rhodopsin interactions, but changes in the crystal lattice are induced by the addition of different surfactant additives. However, the intermolecular interactions between rhodopsin molecules in these crystal structures may reflect the contacts necessary for the maintenance of dimers or oligomers in rod outer segment membranes. Similar contacts may assist in the formation of dimers or oligomers in other GPCRs as well. These new dimers are compared with other models proposed by crystallography or EM and AFM studies. The inter-monomer surface contacts are different for each model, but several of these models coincide in implicating helix I, II, and H-8 as contributors to the main contact surface stabilizing the dimers.

Interaction of chromophore, 11-cis-retinal, with amino acid residues of the visual pigment rhodopsin in the region of protonated Schiff base: A molecular dynamics study

A molecular dynamics study of the dark adapted visual pigment rhodopsin molecule was carried out. The interaction of the chromophore group, 11-cis-retinal, with the nearest amino acid residues in the chromophore center of the molecule, namely, in the region of the protonated Schiff base linkage, was analyzed. Most likely, the interaction of the CH=NH bond with the negatively charged amino acid residue Glu113 cannot be described as a simple electrostatic interaction of two oppositely charged groups. One can propose that not only Glu113 but also Glu181 and Ser186 are involved in stabilization of the protonated Schiff base linkage. Accord-ing to calculations, Glu181 interacts, as the counter-ion, with the Schiff base indirectly via Ser186. The intramolecular mechanisms of protonated Schiff base stabilization in rhodopsin are discussed.

Seeing Is Believing

In order for vertebrate photoreceptors to exhibit their exquisite sensitivity that allows them to distinguish stimulation by a single photon, the sensor, rhodopsin, must have a very reproducible response. Rhodopsin propagates the signal from absorbed photons by activating transducin, but it is then inactivated by phosphorylation. Doan et al. measured the response of single rhodopsin molecules to single-photon absorption events in preparations of mouse photoreceptors. Multiple phosphorylation events provide independent inhibitory signals that together may provide the remarkable reproducibility of the amplitude and duration of rhodopsin activation observed in the vertebrate eye. T. Doan, A. Mendez, P. B. Detwiler, J. Chen, F. Rieke, Multiple phosphorylation sites confer reproducibility of the rod's single-photon responses. Science 313 , 530-533 (2006). [Abstract] [Full Text]

Frequency up-conversion of infrared laser radiation in the human retina

Experimental observation is reported of conversion in a human retina of infrared laser radiation (with a wavelength of 1.06 μm) into the visible upon scattering by a solid target. The wavelength of this visible radiation, as estimated by several independent observers, is around 0.557 μm. Frequency up-conversion is observed down to a peak power on the retina of only 150 W/cm2, while, for other laser sources, this threshold is as low as 1 W/cm2. It is suggested that the observed conversion is due to the second-harmonic generation in the periodic structure of the retina of a human eye. Deviation of the observed wavelength from that of the second harmonic in vacuum is ascribed to the spectral dependence of the refractive index of rhodopsin molecules within the retina.

Functional and Structural Characterization of Rhodopsin Oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS)

Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.

Visual pigment rhodopsin : a computer simulation of the molecular dynamics of 11- cis-retinal chromophore and amino-acid residues in the chromophore centre

Based on a computer molecular simulation, we have investigated the conformational dynamics of rhodopsin and its free opsin. A special emphasis was made on the behaviour of the chromophore group – 11-cis-retinal within the rhodopsin molecule in its dark-adapted state. The molecular dynamics trajectories were traced in the time range of 3000 ps. We have generated 3×106 discrete states of free opsin and rhodopsin in order to compare the rhodopsin and opsin structural conformation changes. Analysis of the 11-cis-retinal adjustment process in the chromophore site of opsin in correlation with the behaviour of the nearest surrounding amino acid residues has been carried out. The possible molecular mechanisms of the conformational adaptation of 11-cis-retinal in the protein binding pocket, which occur during the physiological regeneration of the visual pigment rhodopsin, are discussed.

Arrestin Interaction With Rhodopsin: Conceptual Models

It is becoming increasingly apparent that G protein-coupled receptors (GPCRs) can exist and function as oligomers. This notion differs from the classical view of signaling wherein the receptor has been presumed to be monomeric. Despite this shift in views, the interpretation of data related to GPCR function is still largely carried out within the framework of a monomeric receptor. Rhodopsin is a prototypical GPCR that initiates phototransduction. Like other GPCRs, the activity of rhodopsin is regulated by phosphorylation and the binding of arrestin. In the current investigation, we have explored by modeling methods the interaction of rhodopsin and arrestin under the assumption that either one or two rhodopsin molecules bind each arrestin molecule. The dimeric receptor framework may provide a more accurate representation of the system and is therefore likely to lead to a better and more accurate understanding of GPCR signaling.

A Clockwork Hypothesis: Synaptic Release by Rod Photoreceptors Must Be Regular

We can see at light intensities much lower than an average of one photon per rod photoreceptor, demonstrating that rods must be able to transmit a signal after absorption of a single photon. However, activation of one rhodopsin molecule (Rh*) hyperpolarizes a mammalian rod by just 1 mV. Based on the properties of the voltage-dependent Ca 2+ channel and data on [Ca 2+] in the rod synaptic terminal, the 1 mV hyperpolarization should reduce the rate of release of quanta of neurotransmitter by only ∼20%. If quantal release were Poisson, the distributions of quantal count in the dark and in response to one Rh* would overlap greatly. Depending on the threshold quantal count, the overlap would generate too frequent false positives in the dark, too few true positives in response to one Rh*, or both. Therefore, quantal release must be regular, giving narrower distributions of quantal count that overlap less. We model regular release as an Erlang process, essentially a mechanism that counts many Poisson events before release of a quantum of neurotransmitter. The combination of appropriately narrow distributions of quantal count and a suitable threshold can give few false positives and appropriate (e.g., 35%) efficiency for one Rh*.

Highlights from the Literature

Highlights from the Literature

Smell's different

G protein amplification occurs automatically in an activated rod cell, but not so simply in olfactory cells, according to Vikas Bhandawat, Johannes Reisert, and King-Wai Yau (Johns Hopkins University, Baltimore, MD). Photon activation of a single rhodopsin molecule activates many G proteins, thereby amplifying the signal until rhodopsin is inactivated by phosphorylation. “Based on this one well-studied system, it has generally been assumed that other G protein pathways behave similarly,” says Yau. Now, his group's analyses of single olfactory receptor neurons reveal a low amplification system. An individual odorant-bound receptor exhibited a very low probability of activating even one downstream G protein molecule, as odorant receptor binding was transient—lasting 1 ms or less. “We expect many other ligand-triggered G protein pathways to behave similarly,” says Yau. Olfaction amplification therefore requires increasing the probability of G protein activation. This could be achieved either via many odorant molecules that continuously bind to receptors, or via a large number of receptors, so that odorants at low concentrations will still be able to find a receptor. “When these events are summated across all receptor molecules on the cell, and all cells express the same receptor protein,” says Bhandawat, “this produces substantial signal amplification and therefore high sensitivity in the brain.” Reference: Bhandawat, V., et al. 2005. Science. 308:1931–1934. [PubMed]

Elementary Response of Olfactory Receptor Neurons to Odorants

Signaling by heterotrimeric GTP-binding proteins (G proteins) drives numerous cellular processes. The number of G protein molecules activated by a single membrane receptor is a determinant of signal amplification, although in most cases this parameter remains unknown. In retinal rod photoreceptors, a long-lived photoisomerized rhodopsin molecule activates many G protein molecules (transducins), yielding substantial amplification and a large elementary (single-photon) response, before rhodopsin activity is terminated. Here we report that the elementary response in olfactory transduction is extremely small. A ligand-bound odorant receptor has a low probability of activating even one G protein molecule because the odorant dwell-time is very brief. Thus, signal amplification in olfactory transduction appears fundamentally different from that of phototransduction.

Organization of rhodopsin molecules in native membranes of rod cells–an old theoretical model compared to new experimental data

It has been shown that rhodopsin forms an oligomer in the shape of long double rows of monomers. Because of the importance of rhodopsin as a template for all G protein-coupled receptors, its dimeric, tetrameric and higher-oligomeric structures also provide a useful pattern for similar structures in GPCRs. New experimental data published recently are discussed in the context of a proposed model of the rhodopsin oligomer 1N3M deposited in the protein data bank. The new rhodopsin structure at 2.2 A resolution with all residues resolved as well as an electron cryomicroscopy structure from 2D crystals of rhodopsin are in agreement with the 1N3M model. Accommodation of movement of transmembrane helix VI, regarded as a major event during the activation of rhodopsin, in a steady structure of the oligomer is also discussed. [Figure: see text]. Superimposition of the 1U19 (red wire), 1GZM (purple wire) and 1N3M (blue wire) rhodopsin structures. Size of the wires is proportional to thermal factors of backbone C(alpha) atoms, view parallel to the membrane.

From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG

To quantify the rate at which light in a ganzfeld produces photoisomerizations in mouse rods in situ, we measured the rate of rhodopsin bleaching in eyes of recently euthanized mice with fully dilated pupils. The amount of rhodopsin declined as a first-order (exponential) function of the duration of the exposure at the luminance of 920 scot cd m −2: the rate constants of bleaching were 8.3 × 10 −6 and 2.8 × 10 −5 s −1 (scot cd −1 m 2) −1 for C57B1/6 and 129P3/J mice, respectively. When the ∼3-fold difference in effective areas of the pupils of the mice are taken into consideration, the bleaching rates for both strains become essentially the same, 2.6 × 10 −6 fraction rhodopsin (scot Td s) −1. Assuming 7 × 10 7 rhodopsin molecules per rod, this bleaching rate yields the result that a flash of 1 scot Td s produces 181 photoisomerizations per rod, a value close to that derived from analysis of the collecting area of the rod for axially propagating light. We measured the electroretinograms of mice of the two strains reared under controlled illumination conditions (2 and 100 lux), and compared their properties, using the calibrations to determine the absolute sensitivities of the b-wave and a-waves. The intensity that produces a half-saturating rod b-wave response is 0.3–0.6 photoisomerizations rod −1, and the amplification constant of the rod a-wave is 5–6 s −2 photoisomerization −1, with little dependence on the strain.