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Crystallization and crystal properties of squid rhodopsin.

ABSTRACT: Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, hexagonal crystals grown in the presence of octylglucoside and ammonium sulfate diffracted to 2.8 A resolution. The diffraction data suggested that the crystal belongs to space group P6(2), with unit-cell parameters a = b = 122.1, c = 158.6 A. Preliminary crystallographic analysis, together with linear dichroism results, suggested that the rhodopsin dimers are packed in such a manner that their transmembrane helices are aligned nearly parallel to the c axis.

SUBMITTER: Murakami M

PROVIDER: S-EPMC2335087 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Crystallization and crystal properties of squid rhodopsin.

Murakami Midori M Kitahara Rei R Gotoh Toshiaki T Kouyama Tsutomu T

Acta crystallographica. Section F, Structural biology and crystallization communications 20070505 Pt 6

Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, he ...[more]

PMID: 17554166

Similar Datasets

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region.

Project description:G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.

| S-EPMC2440622 | biostudies-literature

Crystallographic Study of the LUMI Intermediate of Squid Rhodopsin.

Project description:Upon absorption of light, the retinal chromophore in rhodopsin isomerizes from the 11-cis to the trans configuration, initiating a photoreaction cycle. The primary photoreaction state, bathorhodopsin (BATHO), relaxes thermally through lumirhodopsin (LUMI) into a photoactive state, metarhodopsin (META), which stimulates the conjugated G-protein. Previous crystallographic studies of squid and bovine rhodopsins have shown that the structural change in the primary photoreaction of squid rhodopsin is considerably different from that observed in bovine rhodopsin. It would be expected that there is a fundamental difference in the subsequent thermal relaxation process between vertebrate and invertebrate rhodopsins. In this work, we performed crystallographic analyses of the LUMI state of squid rhodopsin using the P62 crystal. When the crystal was illuminated at 100 K with blue light, a half fraction of the protein was converted into BATHO. This reaction state relaxed into LUMI when the illuminated crystal was warmed in the dark to 170 K. It was found that, whereas trans retinal is largely twisted in BATHO, it takes on a more planar configuration in LUMI. This relaxation of retinal is accompanied by reorientation of the Schiff base NH bond, the hydrogen-bonding partner of which is switched to Asn185 in LUMI. Unlike bovine rhodopsin, the BATHO-to-LUMI transition in squid rhodopsin was accompanied by no significant change in the position/orientation of the beta-ionone ring of retinal.

| S-EPMC4449009 | biostudies-literature

Coupling of retinal, protein, and water dynamics in squid rhodopsin.

Project description:The light-induced isomerization of the retinal from 11-cis to all-trans triggers changes in the conformation of visual rhodopsins that lead to the formation of the activated state, which is ready to interact with the G protein. To begin to understand how changes in the structure and dynamics of the retinal are transmitted to the protein, we performed molecular dynamics simulations of squid rhodopsin with 11-cis and all-trans retinal, and with two different force fields for describing the retinal molecule. The results indicate that structural rearrangements in the binding pocket, albeit small, propagate toward the cytoplasmic side of the protein, and affect the dynamics of internal water molecules. The sensitivity of the active-site interactions on the retinal force-field parameters highlights the coupling between the retinal molecule and its immediate protein environment.

| S-EPMC3042571 | biostudies-literature

Crystallization, X-ray diffraction analysis and SIRAS/molecular-replacenent phasing of three crystal forms of Anabaena sensory rhodopsin transducer.

Project description:Anabaena sensory rhodopsin transducer (ASRT) is a 14.7 kDa soluble signaling protein associated with the membrane-embedded light receptor Anabaena sensory rhodopsin (ASR) from Anabaena sp., a freshwater cyanobacterium. Crystals of ASRT were obtained in three different space groups, P4, C2 and P2(1)2(1)2(1), which diffract to 1.8, 2.1 and 2.0 angstroms, respectively. Phases for one of these crystal forms (P4) were obtained by SIRAS phasing using an iodide quick-soak derivative and a partial model was built. Phases for the remaining crystal forms were obtained by molecular replacement using the partial model from the P4 crystal form.

| S-EPMC2222576 | biostudies-literature

Crystal packing analysis of Rhodopsin crystals.

Project description: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.

| S-EPMC1950280 | biostudies-literature

Activation of the GTP-binding protein Gq by rhodopsin in squid photoreceptors.

Project description:Photoaffinity labelling by a GTP analogue has been used to identify a 42 kDa band as the major G alpha subunit in squid photoreceptor membranes, recently identified by partial sequence analysis to be a member of the Gq sub-group of GTP-binding proteins [Pottinger, Ryba, Keen & Findlay (1991) Biochem. J. 279, 323-326]. Guanine-nucleotide-binding displacement analysis gave a stoichiometry of 1 G-protein per 12.5 rhodopsin molecules, the same as in vertebrate rod photoreceptors. Binding was not detected above background in the dark, but was rapidly activated by light. Unlike vertebrate transducin, this G-protein is very temperature-sensitive. GTP binding is maximal at temperatures less than 10 degrees C and is much decreased after several minutes above 18 degrees C. The light-stimulated GTPase rate is maximal around 10 degrees C, above which the loss of binding sites counteracts the increase in hydrolytic rate per site. Earlier studies described light-sensitive G alpha components of 40 and 45 kDa, by ADP-ribosylation in the presence of cholera and pertussis toxins. These are now shown to be very minor components, as the prolonged treatment at elevated temperature required for ADP-ribosylation is sufficient to inactivate the major G alpha totally. Unlike the minor G alpha components, the 42 kDa G alpha is not inhibited by Ca2+.

| S-EPMC1133198 | biostudies-other

Purification, crystallization and preliminary X-ray crystallographic analysis of squid heavy meromyosin.

Project description:All muscle-based movement is dependent upon carefully choreographed interactions between the two major muscle components, myosin and actin. Regulation of vertebrate smooth and molluscan muscle contraction is myosin based (both are in the myosin II class), and requires the double-headed form of myosin. Removal of Ca2+ from these muscles promotes a relatively compact conformation of the myosin dimer, which inhibits its interaction with actin. Although atomic structures of single myosin heads are available, the structure of any double-headed portion of myosin, including the ∼375 kDa heavy meromyosin (HMM), has only been visualized at low (∼20 Å) resolution by electron microscopy. Here, the growth of three-dimensional crystals of HMM with near-atomic resolution (up to ∼5 Å) and their X-ray diffraction are reported for the first time. These crystals were grown in off-state conditions, that is in the absence of Ca2+ and the presence of nucleotide analogs, using HMM from the funnel retractor muscle of squid. In addition to the crystallization conditions, the techniques used to isolate and purify this HMM are also described. Efforts at phasing and improving the resolution of the data in order to determine the structure are ongoing.

| S-EPMC3606567 | biostudies-literature

Squid

Project description:A Molecular Tandem-Repeat Strategy For Elucidating Mechanical Properties of High Strength Proteins

| PRJNA320263 | ENA

Crystal structure of the Anabaena sensory rhodopsin transducer.

Project description:We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.

| S-EPMC2041937 | biostudies-literature

Crystal structure of a thermally stable rhodopsin mutant.

Project description:We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 microm X-ray beam were used to collect data along a single needle measuring 5 microm x 5 microm x 90 microm. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.

| S-EPMC2258155 | biostudies-literature

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