Rod Outer Segment Disk Research Articles

Impact of reduced rhodopsin expression on the structure of rod outer segment disc membranes.

Rhodopsin is the light receptor embedded in rod outer segment (ROS) disc membranes of photoreceptor cells that initiates vision via phototransduction. The relationship between rhodopsin expression and the formation of membrane structures in the ROS is unclear but important to better understand both normal function and pathological conditions. To determine the impact of reduced rhodopsin expression on the structure of ROS discs and the supramolecular organization of rhodopsin, ROS disc membrane samples from heterozygous rhodopsin knockout mice were examined by atomic force microscopy. Similar to rhodopsin in wild-type mice, rhodopsin formed nanodomains in ROS disc membranes of heterozygous knockout mice. The reduced rhodopsin expression in heterozygous knockout mice resulted in ROS disc membranes that were smaller compared to those in wild-type mice at all ages tested. Changes in ROS disc membrane properties were observed between 4 and 6 weeks of age in heterozygous knockout mice that were not present in age-matched wild-type mice. In 4 week old mice, the number and density of rhodopsin in ROS disc membranes was lower than that in age-matched wild-type mice. In contrast, 6 and 8 week old mice had more rhodopsin molecules present in disc membranes compared to 4 week old mice, which resulted in rhodopsin densities similar to those found in age-matched wild-type mice. Thus, mechanisms appear to be present that maintain a constant density of rhodopsin within ROS disc membranes even when reducing the expression of the light receptor by about half. These adaptive mechanisms, however, only occur after 4 weeks of age.

Exclusion of the unfolded protein response in light-induced retinal degeneration in the canine T4R RHO model of autosomal dominant retinitis pigmentosa.

PurposeTo examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHO dog).MethodsThe left eyes of T4R RHO dogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure.ResultsA brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3.ConclusionThe UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration.

Insights into the Molecular Properties of ABCA4 and Its Role in the Visual Cycle and Stargardt Disease.

ABCA4 is a member of the A-subfamily of ATP-binding cassette (ABC) transporters localized in rod and cone outer segment disc membranes. Over 800 mutations in ABCA4 are now known to cause Stargardt macular degeneration and related retinal degenerative diseases. Biochemical studies have shown that ABCA4 transports or flips the 11-cis and all-trans isomers of N-retinylidene-phosphatidylethanolamine across disc membranes, thereby facilitating the removal of retinal from disc membranes through the visual cycle and preventing the accumulation of potentially toxic bisretinoid compounds in photoreceptor and retinal pigment epithelial cells. This chapter summarizes studies which have led to our understanding of the role of ABCA4 in the visual cycle and Stargardt disease.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Nanodomain organization of rhodopsin in native human and murine rod outer segment disc membranes

Biological membranes display distinct domains that organize membrane proteins and signaling molecules to facilitate efficient and reliable signaling. The organization of rhodopsin, a G protein-coupled receptor, in native rod outer segment disc membranes was investigated by atomic force microscopy. Atomic force microscopy revealed that rhodopsin is arranged into domains of variable size, which we refer to herein as nanodomains, in native membranes. Quantitative analysis of 150 disc membranes revealed that the physical properties of nanodomains are conserved in humans and mice and that the properties of individual disc membranes can be variable. Examining the variable properties of disc membranes revealed some of the factors contributing to the size of rod outer segment discs and the formation of nanodomains in the membrane. The diameter of rod outer segment discs was dependent on the number of rhodopsin molecules incorporated into the membrane but independent of the spatial density of rhodopsin. The number of nanodomains present in a single disc was also dependent on the number of rhodopsin molecules incorporated into the membrane. The size of the nanodomains was largely independent of the number or spatial density of rhodopsin in the membrane.

Initiation of rod outer segment disc formation requires RDS.

Rod outer segment (OS) morphogenesis involves assembly of flattened discs circumscribed by a hairpin-like rim, however, the role of the rim and rim proteins such as retinal degeneration slow (RDS) and its homologue rod OS membrane protein-1 (ROM-1) in this process remains unclear. Here we show that without RDS, no disc/OS formation occurs, while without rhodopsin, small OS structures form containing aligned nascent discs. In the absence of both rhodopsin and RDS, RDS-associated degeneration is slowed, and ROM-1 is stabilized and trafficked to the OS. These animals (rho − /− /rds − /−) exhibit OSs slightly better than those lacking only RDS, but still without signs of disc formation. These results clearly demonstrate that OS morphogenesis is initiated by RDS-mediated rim formation, a process ROM-1 cannot recapitulate, with subsequent disc growth mediated by rhodopsin. The critical role of RDS in this process helps explain why photoreceptors are so sensitive to varied RDS levels, and why mutations in RDS cause debilitating retinal disease.

Mutant ELOVL4 that causes autosomal dominant stargardt-3 macular dystrophy is misrouted to rod outer segment disks.

Autosomal dominant Stargardt macular dystrophy caused by mutations in the Elongation of Very Long Chain fatty acids (ELOVL4) gene results in macular degeneration, leading to early childhood blindness. Transgenic mice and pigs expressing mutant ELOVL4 develop progressive photoreceptor degeneration. The mechanism by which these mutations cause macular degeneration remains unclear, but have been hypothesized to involve the loss of an ER-retention dilysine motif located in the extreme C-terminus. Dominant negative mechanisms and reduction in retinal polyunsaturated fatty acids also have been suggested. To understand the molecular mechanisms involved in disease progression in vivo, we addressed the hypothesis that the disease-linked C-terminal truncation mutant of ELOVL4 exerts a dominant negative effect on wild-type (WT) ELOVL4, altering its subcellular localization and function, which subsequently induces retinal degeneration and loss of vision. We generated transgenic Xenopus laevis that overexpress HA-tagged murine ELOVL4 variants in rod photoreceptors. Tagged or untagged WT ELOVL4 localized primarily to inner segments. However, the mutant protein lacking the dilysine motif was mislocalized to post-Golgi compartments and outer segment disks. Coexpression of mutant and WT ELOVL4 in rods did not result in mislocalization of the WT protein to outer segments or in the formation of aggregates. Full-length HA-tagged ELOVL4 lacking the dilysine motif (K308R/K310R) necessary for targeting the WT ELOVL4 protein to the endoplasmic reticulum was similarly mislocalized to outer segments. We propose that expression and outer segment mislocalization of the disease-linked 5-base-pair deletion mutant ELOVL4 protein alters photoreceptor structure and function, which subsequently results in retinal degeneration, and suggest three possible mechanisms by which mutant ELOVL4 may induce retinal degeneration in STGD3.

Rhodopsin Crowding in Model Lipid Bilayers - Functional Implications

In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. It has been hotly debated if oligomerizationof rhodopsin is a critical step for efficient activation of G-protein. Here, we investigated the effect of rhodopsin density in synthetic membranes on the interaction with its cognate G-protein transducin (Gt). Experiments were conducted at rhodopsin:lipid ratios ranging from 1:4000 to 1:70 with sn1-stearoyl sn2-oleoyl phosphatidylcholine (18:0-18:1 PC) model bilayers at ambient temperature.The amount of metarhodopsin-II (MII) formed after photoactivation was determined by UV-visible spectroscopy. Guanine nucleotide exchange rate measured using labeled GTPγS was used to monitor Gt affinity for activated rhodopsin, the rate of rhodopsin catalyzed Gt activation, and the decay rate of the active photointermediate.At low rhodopsin density (1:1000 and below), MII concentration was the highest and independent of rhodopsin concentration. Increasing rhodopsin packing density correlated with a shift in the metarhodopsin-I (MI)/MII equilibrium towards MI. After photoactivation, rhodopsin decayed at two different rates (t1/2 ∼ 3 min and > 60 min) and the proportion of the fast decaying photointermediate decreased with increasing rhodopsin density. Finally, MII-catalyzed GDP-GTPγS nucleotide exchange rates for Gt were crucially affected by rhodopsin density. The enzymatic power of rhodopsin (Vmax/Km for Gt) was higher by 2 orders of magnitude at low rhodopsin density as compared to high rhodopsin density.Our results suggest that, in model membranes, rhodopsin exists as an equilibrium between at least two populations: monomeric at low rhodopsin density with rapid decay and high catalytic efficiency and oligomeric at high rhodopsin density with slow decay and inefficient catalysis of nucleotide exchange.

Functional expression of oxidative phosphorylation proteins in the rod outer segment disc

The rod Outer Segment (OS) disc, an organelle devoid of mitochondria, is specialized in phototransduction, a process requiring a continual chemical energy supply. We have shown that OS discs express functional mitochondrial electron transport chains, Fo F1 -ATP synthase and the tricarboxylic acid cycle enzymes, all mitochondrial features. Here, we focus on oxygen consumption and adenosine triphosphate (ATP) synthesis by OS discs analysing electron transport chain I-III-IV and II-II-IV pathways, supported by reduced nicotinamide adenine dinucleotide and succinate, respectively. Interestingly, respiratory capacity of discs was measurable also in the presence of 3-hydroxy-butyrrate, a typical metabolic substrate for the brain. Data were supported by a two-dimensional electrophoresis analyses conducted as our previous one, but focused to those mitochondrial proteins that are involved in oxidative phosphorylation. Carbonic anhydrase was also found active in OS discs. Moreover, colocalization of Rhodopsin with respiratory complex I and ATP synthase seems a further step in the characterization of some proteins typical of the mitochondrial inner membranes that are expressed in the rod discs. The existence of oxygen utilization in the outer retina, likely supplying ATP for phototransduction, may shed light on some retinal pathologies related to oxidative stress of the outer retina.

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.

Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Prominin-1 Localizes to the Open Rims of Outer Segment Lamellae inXenopus laevisRod and Cone Photoreceptors

Prominin-1 expresses in rod and cone photoreceptors. Mutations in the prominin-1 gene cause retinal degeneration in humans. In this study, the authors investigated the expression and subcellular localization of xlProminin-1 protein, the Xenopus laevis ortholog of prominin-1, in rod and cone photoreceptors of this frog. Antibodies specific for xlProminin-1 were generated. Immunoblotting was used to study the expression and posttranslational processing of xlProminin-1 protein. Immunocytochemical light and electron microscopy and transgenesis were used to study the subcellular distribution of xlProminin-1. xlProminin-1 is expressed and is subject to posttranslational proteolytic processing in the retina, brain, and kidney. xlProminin-1 is differently expressed and localized in outer segments of rod and cone photoreceptors of X. laevis. Antibodies specific for the N or C termini of xlProminin-1 labeled the open rims of lamellae of cone outer segments (COS) and the open lamellae at the base of rod outer segments (ROS). By contrast, anti-peripherin-2/rds antibody, Xper5A11, labeled the closed rims of cone lamellae adjacent to the ciliary axoneme and the rims of the closed ROS disks. The extent of labeling of the basal ROS by anti-xlProminin-1 antibodies varied with the light cycle in this frog. The entire ROS was also faintly labeled by both antibodies, a result that contrasts with the current notion that prominin-1 localizes only to the basal ROS. These findings suggest that xlProminin-1 may serve as an anti-fusogenic factor in the regulation of disk morphogenesis and may help to maintain the open lamellar structure of basal ROS and COS disks in X. laevis photoreceptors.

Cholesterol Enhances or Reduces Metarhodopsin II Formation Depending on Bilayer Thickness

Cholesterol is one of the most efficient modulators of G Protein-Coupled Receptor (GPCR) function. We monitored the effect of cholesterol on rhodopsin function for a set of bilayers with different hydrophobic thickness. Surprisingly, cholesterol shifts the Metarhodopsin-I (MI)/Metarhodopsin-II (MII) equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (2.7 nm), and to MI for thicker bilayers. In previous work conducted on rod outer segment disks and model membranes, increasing cholesterol concentration always shifted the equilibrium towards MI. It was proposed that the cholesterol effect is primarily related to a tighter packing of lipid hydrocarbon chains which generates a less permissive environment for the formation of MII. To gain deeper insights into mechanisms, we followed changes in lipid-rhodopsin interaction by 2H NMR using deuterated lipids. It was reported by us and the Brown laboratory that an increase of bilayer hydrophobic thickness in the absence of cholesterol favors MII with a turnover to MI for bilayers that are very thick. Indeed, the cholesterol-induced shifts towards MII for thinner membranes correlated nicely with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR suggesting that the increase in bilayer thickness by cholesterol plays a major role in controlling the energetics of the MI-MII equilibrium. Furthermore, changes in average lipid order parameters due to the presence of rhodopsin were much larger in cholesterol-containing membranes than in cholesterol-free membranes suggesting strongly that the perturbation in the lipid matrix from protein insertion reaches much further away from the protein. This is expected for membranes that are stiffer due to the presence of cholesterol. The consequences of these findings for lipid mediated shifts in rhodopsin function and rhodopsin-rhodopsin interactions will be discussed.

Lipid and Detergent Effects on the Kinetic Stability of the GPCR, Rhodopsin

The photoreceptor, rhodopsin is a kinetically stable G-Protein Coupled Receptor that is located in rod outer segment disk membranes. Differential scanning calorimetry (DSC) studies have shown that rhodopsin exhibits an irreversible scan rate dependent endothermic transition (Tm) at approximately 72 oC. The activation energy for thermal denaturation (Eact) calculated from the scan rate dependence of the Tm is sensitive to the integrity of the lipid bilayer. It is also sensitive to proteolytic cleavage of the extramembraneous loops of rhodopsin. Here we investigate the influence of palmitoylation, membrane cholesterol and digitonin solubilization on rhodopsin kinetic stability. DSC experiments were performed using a MicroCal VP-DSC microcalorimeter. Samples were scanned at 15, 30, 60 and 90o/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. Rhodopsin palmitoylation at cys 322 and cys 323 anchors the C-terminus to the membrane. The Eact for rhodopsin in disk membranes treated with hydroxylamine to remove these palmitate groups was approximately 35 Kcal/mole less than that of native rhodopsin. The Eact for rhodopsin was also determined at different membrane cholesterol levels. These data indicated a decrease in stability both above and below native membrane cholesterol levels. Finally, unlike solubilization in other detergents, solubilization in digitonin had little effect on rhodopsin kinetic stability.

Biochemical Crosslinking and Liquid Chromatography-Mass Spectrometry Demonstrate a Rhodopsin Dimerization Interface Mediated by Helices 1 and 8

Heptahelical G protein-coupled receptors (GPCRs) can exist as monomers and a ternary complex of ligand, GPCR and heterotrimeric G protein is the basic signaling unit. GPCRs also tend to form dimers and higher-order oligomers in membranes, although the functional consequences of these interactions are in most cases unknown. Furthermore, the precise protein-protein interface(s) in receptor dimers and the mechanisms mediating dimerization remain controversial. Two-dimensional and three-dimensional densities obtained from electron microscopy, as well as X-ray data on packing of rhodopsin crystals, suggest that the primary dimer contacts involve transmembrane helix (H) 1 and cytoplasmic H8. Biochemical crosslinking studies with dopamine D2 receptors hint that this interface may be broadly significant. We crosslinked rhodopsin dimers in native rod outer segment disk membranes to demonstrate the proximity of H8 between adjacent receptors. Four homobifunctional cysteine-reactive crosslinkers were used, two with maleimide groups and two with methanethiosulfonate groups. The formation of dimers and oligomers was verified by SDS-PAGE, size-exclusion chromatography, and immunoblot analysis. We used partial proteolysis and high-resolution liquid chromatography-mass spectrometry (LC-MS) to identify the site of a crosslink between Cys316-Cys316. Cys316 is one of two reactive cysteines in rhodopsin and is located in H8. The spacer length of the crosslinkers that formed the intermolecular Cys316-Cys316 crosslink is consistent with the distance predicted in a H1/H8 dimer model. This result corroborates findings from coarse-grained molecular dynamics (CGMD) potential of mean force (PMF) calculations, which show that the H1/H8 orientation is by far the most stable among the possible dimer orientations tested. Together these results strongly suggest the existence of this interface in native membranes. Given the high degree of homology across class A GPCRs, these results may be relevant for other receptors.

Targeting of mouse guanylate cyclase 1 ( Gucy2e) to Xenopus laevis rod outer segments

Photoreceptor guanylate cyclase (GC1) is a transmembrane protein and responsible for synthesis of cGMP, the secondary messenger of phototransduction. It consists of an extracellular domain, a single transmembrane domain, and an intracellular domain. It is unknown how GC1 targets to the outer segments where it resides. To identify a putative GC1 targeting signal, we generated a series of peripheral membrane and transmembrane constructs encoding extracellular and intracellular mouse GC1 fragments fused to EGFP. The constructs were expressed in Xenopus laevis rod photoreceptors under the control of the rhodopsin promoter. We examined the localization of GFP-GC1 fusion proteins containing the complete GC1 sequence, or partial GC1 sequences, which were membrane-associated via either the GC1 transmembrane domain or the rhodopsin C-terminal palmitoyl chains. Full-length GFP-GC1 targeted to the rod outer segment disk rims. As a group, fusion proteins containing the entire cytoplasmic domain of GC1 targeted to the OS, whereas other fusion proteins containing portions of the cytoplasmic or the extracellular domains did not. We conclude that GC1 likely has no single linear peptide-based OS targeting signal. Our results suggest targeting is due to either multiple weak signals in the cytoplasmic domain of GC1, or co-transport to the OS with an accessory protein.

The Bilayer Enhances Rhodopsin Kinetic Stability in Bovine Rod Outer Segment Disk Membranes

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl- β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl- β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature ( T m) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T m as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation ( E act) was calculated from the scan rate dependence of the T m and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E act as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E act to rhodopsin denaturation.

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26 000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.

Peripherin/rds co-distributes with putative binding partners in basal rod outer segment disks

The retinal degeneration slow protein peripherin/rds, (a.k.a. RDS), encoded by the rds gene, is essential for the formation, organization and maintenance of outer segments (Arikawa et al., 1992; Connell et al., 1991; Connell and Molday, 1990; Kedzierski et al., 2001; Molday, 1998). In humans, any one of over 160 mutations within this gene results in a broad variety of late onset progressive retinal dystrophies characterized by abnormal photoreceptor structure (Dryja et al., 1997; Keen and Inglehearn, 1996; Kohl et al., 1998). The pathogenic mechanism(s) underlying peripherin/rds mediated retinal dystrophies are unknown, however new insight has been gained in our understanding of peripherin/rds function based upon recent structural studies identifying the C-terminal domain of this protein as homologous to structures called intrinsically disordered domains (IDD) (Dyson, 2005; Edrington, 2007b; Ritter, 2005). IDDs are unstructured domains that become structurally stabilized by interacting with one or with several binding partners (Uversky et al., 2008). Structural flexibility imparts multi-functionality to the protein and binding partners act as regulators of its function (Lakoucheva et al., 2002; Vucetic et al., 2007). In humans, numerous mutations in the rds C-terminus result in rod/cone or cone/rod dystrophies. In a murine model of retinitis pigmentosa, deletion of codon 307 in the rds gene results in severely dysmorphic outer segments (OSs) in mice (McNally et al., 2002). The rds 307 del mouse provides the first in vivo evidence for a correlation between intrinsic disorder and retinopathy. In these mice disruption of the peripherin/rds C-terminus results in a gene product with a more intrinsically ordered C-terminal domain characterized by an increase in α-helical content.

Rhodopsin - Rhodopsin Oligomerization in Model Lipid Bilayers - Functional Implications

We studied rhodopsin oligomerization as a function of rhodopsin concentration and lipid composition and related oligomerization to shifts in rhodopsin function. In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. In model membranes, increasing rhodopsin packing density was linked to a shift in the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium towards MI as well as to lower rates of MII formation. We reconstituted rhodopsin into various phosphatidylcholine bilayers at rhodopsin/lipid ratios ranging from 1:1,000 to 1:70 and followed rhodopsin oligomerization by cross linking of rhodopsin and changes in lipid-rhodopsin interactions by 2H NMR. The amount of MII formed after photoactivation was determined by UV/vis spectroscopy and the rate of transducin activation studied with a GTPγS-assay. At low rhodopsin concentrations (1/300 and lower) rhodopsin appears to be predominantly monomeric. At rhodopsin/lipid ratios higher than 1/300, the level of oligomerization increases in a highly cooperative fashion with concentration such that at physiological concentrations rhodopsin is mostly oligomeric. Protein function correlated tightly with rhodopsin oligomerization. Data on the influence of bilayer properties on the monomer - oligomer transition of rhodopsin and the rate of transducin activation will be presented.