Characterisation of an improved two-dimensional p22121 crystal from bovine rhodopsin

Krebs, Angelika; Villa, Claudio; Edwards, Patricia C.; Schertler, Gebhard F. X.

doi:10.1006/jmbi.1998.2070

Cited by 100 publications

(74 citation statements)

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“…Achieving this goal requires both the determination of the photointermediate sequence under physiological conditions by methods with high time resolution and the application of structural tools that are likely to require nonphysiological constraints. The work reported here sought to characterize the intermediates formed in rhodopsin 2D crystals, a phase which has been of great importance in rhodopsin structure determination (12) and which was used to determine the first structure of a rhodopsin photointermediate (8). Since 2D crystals are essentially similar to the membrane phase, it is reasonable that there would be similarity in the mechanistic scheme followed in those two environments, and to a first approximation the bleaching kinetics in rhodopsin crystal suspensions can be described by a subset of the mechanism that prevails in membrane suspensions of rhodopsin (20) : where in the mechanism above, the italicized part does not appreciably take place on the time scale studied here in crystal suspensions in the 15 − 35 °C range of temperatures, and the abbreviation NRO stands for n-retinylidene opsin, the mixture of non-specific retinal Schiff bases referred to earlier.…”

Section: Discussionmentioning

confidence: 99%

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Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures

et al. 2006

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Bovine rhodopsin photointermediates formed in 2D rhodopsin crystal suspensions were studied by measuring the time dependent absorbance changes produced after excitation with 7 nanosecond laser pulses at 15, 25 and 35 °C. The crystalline environment favored the Meta I 480 photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 °C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I 480 decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35°C and quenched to 19 °C showed that a rapid equilibrium existed on the ∼1 second time scale which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII a ), forms in 2D rhodopsin crystals, but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.Rhodopsin, the visual pigment used in dim light, originally attracted attention as the majority photoreceptor protein in mammalian retinas, with study of the intermediates in its photoactivation sequence starting long before its function as a G protein-coupled receptor (GPCR) was revealed (1). In virtually all other GPCRs the study of activation intermediates is extremely difficult because the diffusional nature of chemoreception does not allow the rapid triggering required for early intermediate characterization using ensemble based methods. While progress has been made in detecting intermediates in chemoreceptor GPCRs (2-4), it is only with great effort that processes 10,000 times slower than those reported here are studied. The detailed structural information available for the inactive, dark state of rhodopsin (5,6) provides an unambiguous basis for structural modeling of the earliest photointermediates. Optical methods of detection, such as time-resolved absorbance measurements (7), provide the highest time resolution of any experimen...

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Section: Discussionmentioning

confidence: 99%

“…Single layer membranes (s), collapsed tubular membranes (t), and protein aggregates (p) are visible. The single layers and the tubes are twodimensional crystals of bovine rhodopsin, which are well characterised (8,12,13). The exact nature of the protein aggregates (p) is not known.…”

Section: Discussionmentioning

confidence: 99%

Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures

et al. 2006

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“…Ref. 51). Indeed, in these crystals the packing of the protein is much looser than in the smaller unit cell, probably due to the presence of more lipids in the crystal and consequently less protein-protein contacts; the dimer molecule is rotated by approximately 45°with respect to the dimer in the smaller lattice, resulting in a larger distance between dimers in the b direction.…”

Section: Calculation Of a Projection Map Of The B 6 F Complex In The mentioning

confidence: 92%

Conformational Changes in the Cytochromeb 6 f Complex Induced by Inhibitor Binding

Breyton

2000

Journal of Biological Chemistry

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Most energy-transducing membranes contain a bc-type complex such as the cytochrome bc 1 complex or the cytochrome b 6 f complex. Members of this family couple electron transfer and proton translocation across the membrane. They also share some structural homologies, having three redox-active subunits in common: the Rieske iron-sulfur protein, a b-type cytochrome (cytochrome b or b 6 ), and a c-type cytochrome (cytochrome c 1 or f). Moreover, there is sequence homology between cytochrome b 6 , subunit IV, and the Rieske protein of the b 6 f complex, and the N-and C-terminal parts of cytochrome b, and the Rieske protein of the bc 1 complex, respectively. Cytochrome c 1 and cytochrome f, on the other hand, do not share any sequence homology. Spectroscopic and EPR properties of the hemes and of the iron-sulfur cluster are similar, although not identical, and some inhibitors affect both complexes equally (reviewed in Refs. 4 -7).The b 6 f complex is found in the thylakoid membrane of higher plants and algae and in the membrane of some cyanobacteria (for reviews, see . Besides the four high molecular weight subunits mentioned above, the b 6 f complex comprises four smaller subunits, PetG, PetL, PetM, and , that have no direct counterpart in the bc 1 complex. The cytochrome b 6 f is the middle component of the photosynthetic chain, coupling the electron transfer from photosystem II (via plastoquinol) to photosystem I (via plastocyanin or a c-type cytochrome).The most widely accepted mechanism for the bc-type complexes is the so-called "Q-cycle" mechanism proposed by Mitchell (18) and modified by Crofts et al. (19). According to this model, two electron paths are distinguished within the complex as follows: a high potential chain, composed of the iron-sulfur cluster of the Rieske protein and the heme of the c-type heme, and a low potential chain, composed of the two hemes of the b-type cytochrome, the low potential b L and high potential b H hemes. Two quinol/quinone-binding sites, located on opposite sides of the membrane, are also predicted: the Q o site, on the intermembrane space/lumenal side of the membrane, and the Q i site, on the matrix/stromal side of the membrane. A turnover of the Q o site comprises the oxidation of a two-electron carrier quinol, one electron reducing the Rieske protein, which in turn reduces the c-type cytochrome and which ultimately reduces plastocyanin or cytochrome c, and the other electron reducing b L , which in turn is oxidized by b H . These events are associated with the release of two protons in the intermembrane space/ lumen. A second turnover reduces another plastocyanine molecule and places both hemes of the b-type cytochrome in a reduced state. The reduction of a quinone at the Q i site results in the oxidation of the two b hemes and an uptake of two protons from the matrix/stroma. Thus, these complexes contribute to generating the proton gradient that drives ATP synthesis in respiration and photosynthesis.Recently, the structure of several bc 1 complexes from mitochondria have ...

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“…Therefore, to obtain information about the helical packing between adjacent molecules we relied on projection maps generated by cryoelectron microscopy. Electron density projection maps have been obtained for bovine (46,47), frog (48), and squid (19) rhodopsin. Comparisons of the helical orientation and packing of the vertebrate and invertebrate rhodopsins show some interesting differences.…”

Section: Introduction Of Cysteines In the Intracellular Loops And Coomentioning

confidence: 99%

C5a Receptor Oligomerization

Klco

Lassere

Baranski

2003

Journal of Biological Chemistry

108

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G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.G protein-coupled receptors (GPCRs) 1 are an evolutionarily conserved family of transmembrane receptors that are characterized by seven-transmembrane helixes connected by intracellular loops (ICs) and extracellular loops (1, 2). GPCRs mediate a multitude of physiologic responses and serve as common pharmacological targets; more than half of all pharmacological agents that are currently prescribed target GPCRs (3). Receptor activation is accomplished by a remarkably diverse group of ligands to catalyze the exchange of GTP for GDP on the ␣ subunit of heterotrimeric G proteins. This exchange results in the dissociation of the ␣-GTP subunit from the ␤␥ dimer. Both complexes can then activate downstream targets, such as effector enzymes and ion channels (4, 5).Despite numerous structure-function studies of many different GPCRs, a fundamental question remains: Do GPCRs function as monomers or as oligomers? For the ϳ25 members of class C receptors, the answer is clear. These receptors form dimers through specialized structures, such as disulfidebonded extracellular domains (metabotropic glutamate receptors) (6, 7) or coiled-coil interactions of the carboxyl-terminal tails (GABA B receptors) (8). For the more than 500 members of the rhodopsin family of GPCRs and the 60 members of the secretin family of GPCRs, studies now demonstrate that a rapidly increasing number of these GPCRs form oligomers (9, 10). Early work...

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Characterisation of an improved two-dimensional p22121 crystal from bovine rhodopsin

Cited by 100 publications

References 34 publications

Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures

Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures

Conformational Changes in the Cytochromeb 6 f Complex Induced by Inhibitor Binding

C5a Receptor Oligomerization

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