Transducin activation by molecular species of rhodopsin other than metarhodopsin II

Decay of metarhodopsin II was accelerated by hydroxylamine treatment or dark incubation of metarhodopsin II at 30 degrees C. The products thus obtained after decay of metarhodopsin II induced GTPase activity on transducin as well as metarhodopsin II suggesting that rhodopsin could activate transducin after the decay of metarhodopsin II intermediate. After urea-treated bovine rod outer segment membrane was completely bleached, rhodopsin in the membrane was regenerated by the addition of 11-cis retinal at various temperatures between 0 and 37 degrees C. The capacity to induce GTPase activity on transducin and phosphate incorporating capacity catalyzed by rhodopsin kinase were measured on such rhodopsins. The results showed that: (1) Regeneration of alpha band of rhodopsin was complete regardless of regeneration temperature; (2) When regenerated at temperatures below 10 degrees C, rhodopsins induced a GTPase activity on transducin in the dark even after treatment with hydroxylamine, whereas rhodopsins after regeneration at temperatures above 13 degrees C did not; (3) When regenerated at 0 degrees C, rhodopsin was phosphorylated if incubated with rhodopsin kinase and ATP in the dark, whereas the spectrally regenerated rhodopsin at 30 degrees C was not. The complete quenching of functions of photoactivated rhodopsin was achieved by recombination with 11-cis retinal at temperatures above 13 degrees C but not below 10 degrees C suggesting the existence of a low temperature intermediate upon regeneration.     

RECONSTITUTION OF SQUID RHODOPSIN IN RHABDOMAL MEMBRANES

Abstract— Squid opsin which is capable of combining with 11- cis or 9- cis retinal to reconstitute photo-pigment has been prepared by irradiation of rhabdomal membranes with orange light (> 530 nm) in the presence of 0.2 M hydroxylamine. When the irradiation is carried out either at concentrations of hydroxylamine higher than 0.2 M or with light of wavelength shorter than 530 nm, rhodopsin in the membranes is bleached quickly, but the ability of the resultant opsin to form rhodopsin is greatly reduced.
The optimum pH for rhodopsin regeneration in rhabdomal membranes was found to be between 6.5 and 8.5. The rate of regeneration of rhodopsin increases with raising temperature, and at about 20°C it is almost the same as that of isorhodopsin. Even after solubilization in digitonin solution, opsin still preserves the ability to reform rhodopsin.
All- trans retinal can be incorporated into retinochrome-bearing membranes, in which it is isomerized into 11- cis isomer by the photoisomerase activity of retinochrome. Rhabdomal membranes retaining active opsin can take up 11- cis retinal from retinochrome membranes so as to synthesize rhodopsin.     

Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes

We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations-as well as their extensive fluctuations-suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.     

Similar membrane affinity of mono- and Di-S-acylated ras membrane anchors: a new twist in the role of protein lipidation

The functionally required membrane attachment of Ras is achieved through an invariant isoprenylation of a C-terminal Cys, supplemented by further lipid modification of adjacent Cys residues by one (N-ras) or two (H-ras) palmitoyls. However, whether the triply lipidated membrane anchor of H-ras has a higher membrane affinity than its doubly lipidated counterpart, or whether the affinity contribution of the two palmitates and the farnesyl is additive, was not known. To address this issue, we carried out potential of mean force (PMF or free energy profile) calculations on a hexadecylated but nonpalmitoylated anchor (Cys186-HD), hexadecylated and monopalmitoylated anchors (Cys181-monopalmitate and Cys184-monopalmitate), and a nonlipid-modified anchor. We found that the overall insertion free energy follows the trend Cys181/Cys184-bipalmitate (wild type) approximately Cys181-monopalmitate > Cys184-monopalmitate > nonpalmitoylated anchor. Consistent with suggestions from recent cell biological experiments, the computed PMFs, coupled with structural analysis, demonstrate that membrane affinity of the Ras anchor depends on both the hydrophobicity of the palmitate and the prenyl groups and the spacing between them. The data further suggest that while Cys181-palmitate and Cys186-farnesyl together provide sufficient hydrophobic force for tight membrane binding, the palmitoyl at Cys184 is likely designed to serve another, presumably functional, role.     

REVERSIBLE BLEACHING OF Chlamydomonas reinhardtii RHODOPSIN in vivo

Abstract— The effect of hydroxylamine on the phototactic activity of Chlamydomonas reinhardtii was investigated. The following results were obtained: (1) wild type cells, irradiated for 10 min with green light immediately after addition of 1 mM hydroxylamine, showed a 20 min transient loss of phototactic activity, (2) irradiation of cells, preincubated in the dark with 4 mM. hydroxylamine for 30 min, diminished the phototactic sensitivity permanently by more than 100-fold without loss of cell motility. (3) The phototactic sensitivity completely recovered within 3(1 min of the removal of hydroxylamin from carotenoid-containing cells or from carotenoid-negative cells upon addition of 11- cis or all- trans retinal. Our explanation is bleaching of rhodopsin by more than 99% and reconstitution by de novo synthesized or by added retinal.     

NANOSECOND LASER PHOTOLYSIS OF RHODOPSIN AND ISORHODOPSIN

Kinetic and spectral measurements have been carried out on the primary intermediate in the photolysis of rhodopsin and isorhodopsin, initiated by a 457 nm, 6 ns (FWHM) laser pulse. In rhodopsin the kinetic decay of bathorhodopsin was found to be 140 ± 15 ns at 20°C. The decay of bathorhodopsin to lumirhodopsin has an activation energy of 51 ± 4 kJ/mol (12.2 ± 1 kcal/mol). The decay kinetics of bathorhodopsin were found to be the same for rhodopsin in membrane and detergent solubilized suspensions. The kinetic decay of the batho product in the photolysis of isorhodopsin was found to be the same as rhodopsin.
The corrected transient spectrum 50 ns following excitation in rhodopsin has two peaks near 560 and 440 nm. A peak was also observed in isorhodopsin near 550 nm at 50 ns following excitation but no transient was observed in the blue. The 550 nm peak in isorhodopsin has an intensity similar to that in rhodopsin indicating that the quantum yields for the formation of batho products of rhodopsin and isorhodopsin are similar under the irradiation conditions used here. Transient spectra for rhodopsin and isorhodopsin 1 μs following excitation are also different. In isorhodopsin the corrected transient spectrum has a peak at 500 nm, similar to low temperature steady state irradiation spectra. The 1 μs transient spectrum in rhodopsin is more intense than in isorhodopsin and shows a peak at 475 nm.     

Control the Mechanical Properties and Degradation of Poly(Glycerol Sebacate) by Substitution of the Hydroxyl Groups with Palmitates

Mechanical properties and degradation profile are important parameters for the applications of biodegradable polyester such as poly(glycerol sebacate) in biomedical engineering. Here, a strategy is reported to make palmitate functionalized poly(glycerol sebacate) (PPGS) to alter the polymer hydrophobicity, crystallinity, microstructures and thermal properties. The changes of these intrinsic properties impart tunable degradation profiles and mechanical properties to the resultant elastomers depending on the palmitate contents. When the palmitates reach up to 16 mol%, the elastic modulus is tuned from initially 838 ± 55 kPa for the PGS to 333 ± 21 kPa for the PPGS under the same crosslinking conditions. The elastomer undergoes reversible elastic deformations for at least 1000 cycles within 20% strain without failure and shows enhanced elasticity. The polymer degradation is simultaneously inhibited because of the increased hydrophobicity. This strategy is different with other PGS modifications which could form a softer elastomer with less crosslinks but typically lead to a quicker degradation. Because the materials are made from endogenous molecules, they possess good cytocompatibility similar to the PGS control. Although these materials are designed specifically for small arteries, it is expected that they will be useful for other soft tissues too.     

Effect of calcium and zinc carboxylates on the thermal stabilisation of PVC

Metal carboxylate stabilisers are believed to replace labile chlorines in PVC with more stable ester linkages resulting in an increase in the stability of the polymer. In the present work, effects of combinations of stearates, palmitates and laurates respectively of zinc and calcium, in various proportions, on the thermal stability of PVC were studied. Combinations of palmitates and stearates having more than 75 mol% of calcium salt were found to increase the stability of the polymer. The combinations of the three carboxylates showed the following order of stabilising: palmitate > stearate > laurate. This effect is explained in terms of a critical chain length of the n-alkyl group of the carboxylate anion which is most effective in the stabilising process. Highly crystalline, low molecular weight polyethylenes are used as plasticisers for PVC. They were found to have a stabilising effect explained in terms of a dilution effect by the non-polar polyethylenes on the polar interactions in PVC; compatibility of polyethylenes with PVC is the limiting factor in this stabilisation.     

Light-regulated permeability of rhodopsin-phospholipid membrane vesicles.

Abstract—Light absorption by rhodopsin in receptor cell membranes initiates the excitation of the receptor cell. Rhodopsin-phospholipid membrane vesicles were studied to localize initial transduction events. Rhodopsin-phospholipid recombinant membranes are thermally stable and light sensitive and may be chemically regenerated after bleaching in the same manner as receptor cell membranes. Rhodopsin-containing vesicles prepared from unsaturated phosphatidylcholine (PCho) or PCho and phosphatidylethanolaminc display kinetics for the metarhodopsin I to II transition which are comparable to those of receptor cell membranes. NMR spectroscopy was used to examine the permeability of the membrane vesicles to added shift (Eu³⁺) or relaxation reagents (Mn²⁺, Co²⁺). Unexposed rhodopsin-phospholipid vesicles are sealed to ion movement and become permeable after light exposure. Selected ions (Ca²⁺, Mn²⁺, Co²⁺) may be photoreleased from the interior of loaded membrane vesicles. The quantity released is proportional to the initial ionic concentration. The number of ions released/rhodopsin bleached is dependent on the light intensity, and high yields (40–160) of Ca²⁺/rhodopsin bleached are observed at low levels of light bleaching. The present results indicate that rhodopsin spans the phospholipid bilayer membrane, and are consistent with an increase in the permeability of the membrane initiated by light excitation of rhodopsin.     

Characterization of Lamprey Rhodopsin by Isolation from Lamprey Retina and Expression in Mammalian Cells

Abstract— A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11- cis -retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti-bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 m M hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.     

Colorimetric determination of copper, nickel and cobalt in their respective soaps

Summary The metal ion content of heavy metal soaps can be successfully determined colorimetrically using their dilute solutions in pyridine. Measurements with copper and nickel soaps (myristates and palmitates) could be carried out at 675 and 650 nm respectively, those for the corresponding cobalt soaps (myristate and palmitate) at 550 nm.

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Zusammenfassung Der Metallgehalt von Schwermetallseifen kann mit gutem Erfolg colorimetrisch bestimmt werden, wenn man die verdünnte Lösung der Substanz in Pyridin verwendet. Messungen an Kupfer- und Nickelseifen (Myristate und Palmitate) wurden bei 675 bzw. 650 nm, an Kobaltseifen (ebenfalls Myristat und Palmitat) bei 550 nm durchgeführt.

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Thanks are due to Prof. A. R. Kidwai for providing facilities and to the Council of Scientific and Industrial Research, India for the award of a fellowship to one of us (R. H.) to carry out this work.     

Effects of nonionic micelles on the rate of alkaline hydrolysis of N-(2'-methoxyphenyl)phthalimide (1): kinetic and rheometric evidence for a transition from spherical to rodlike micelles under the typical reaction conditions

Pseudo-first-order rate constants (k(obs)) for alkaline hydrolysis of N-(2'-methoxyphenyl)phthalimide (1) decrease nonlinearly with increasing total concentration of nonionic surfactant C(m)E(n) (i.e. [C(m)E(n)](T) where m and n represent the respective number of methyl/methylene units in the tail and polyoxyethylene units in the headgroup of a surfactant molecule and m/n=16/20, 12/23 and 18/20) at constant 2% v/v CH(3)CN and 1.0 mM NaOH. The k(obs)vs. [C(m)E(n)](T) data follow the pseudophase micellar (PM) model at ≤ 50 mM C(16)E(20), ≤ 1.4 mM C(12)E(23) and ≤ 2.0 mM C(18)E(20) where rate of hydrolysis of 1 in micellar pseudophase could not be detected. The values of k(obs) fail to follow the PM model at > ～50 mM C(16)E(20), > ～1.4 mM C(12)E(23) and > ～2.0 mM C(18)E(20) which has been attributed to a micellar structural transition from spherical to rodlike which in turn increases C(m)E(n) micellar binding constant (K(S)) of 1 with increasing values of [C(m)E(n)](T). Rheological measurements show the presence of spherical micelles at ≤ 50 mM C(16)E(20), ≤ 1.4 mM C(12)E(23) and ≤ 3.0 mM C(18)E(20). The presence of rodlike micelles is evident from rheological measurements at > ～50 mM C(16)E(20), > ～1.4 mM C(12)E(23) and > ～3.0 mM C(18)E(20).     

A Usual G‐Protein‐Coupled Receptor in Unusual Membranes

G‐protein‐coupled receptors (GPCRs) are the largest family of membrane‐bound receptors and constitute about 50 % of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge‐interaction‐directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid‐ and polymer‐based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non‐biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H⁺] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin‐conserved ERY sequence motif that helps break the cytoplasmic “ionic lock”. This study unveils a novel design concept of non‐biological membranes to reconstitute and harness GPCR functions in synthetic systems.     

A novel biosensor for specific determination of hydrogen peroxide: catalase enzyme electrode based on dissolved oxygen probe

A biosensor for the specific determination of hydrogen peroxide was developed using catalase (EC 1.11.1.6) in combination with a dissolved oxygen probe. Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane served as enzyme electrode. The electrode response was maximum when 50 mM phosphate buffer was used at pH 7.0 and at 35 degrees C. The biosensor response depends linearly on hydrogen peroxide concentration between 1.0x10(-5) and 3.0x10(-3) M with a response time of 30 s. The sensor is stable for >3 months so in this period >400 assays can be performed.     

Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers

The G protein-coupled receptor (GPCR) rhodopsin self-assembles into supramolecular structures in native bilayers, but the structural determinants of receptor oligomerization are not known. We carried out multiple self-assembly coarse-grained molecular dynamics (CGMD) simulations of model membranes containing up to 64 molecules of the visual receptor rhodopsin over time scales reaching 100 μs. The simulations show strong preferential interaction modes between receptors. Two primary modes of receptor-receptor interactions are consistent with umbrella sampling/potential of mean force (PMF) calculations as a function of the distance between a pair of receptors. The preferential interfaces, involving helices (H) 1/8, 4/5 and 5, present no energy barrier to forming a very stable receptor dimer. Most notably, the PMFs show that the preferred rhodopsin dimer exists in a tail-to-tail conformation, with the interface comprising transmembrane H1/H2 and amphipathic H8 at the extracellular and cytoplasmic surfaces, respectively. This dimer orientation is in line with earlier electron microscopy, X-ray, and cross-linking experiments of rhodopsin and other GPCRs. Less stable interfaces, involving H4 and H6, have a free energy barrier for desolvation (delipidation) of the interfaces and appear to be designed to stabilize "lubricated" (i.e., lipid-coated) dimers. The overall CGMD strategy used here is general and can be applied to study the homo- and heterodimerization of GPCRs and other transmembrane proteins. Systematic extension of the work will deepen our understanding of the forces involved in the membrane organization of integral membrane proteins.     

BUNDLE: A program for building the transmembrane domains of G-protein-coupled receptors

The only information available at present about the structural features of G-protein-coupled receptors (GPCRs) comes from low resolution electron density maps of rhodopsin obtained from electron microscopy studies on 2D crystals. Despite their low resolution, maps can be used to extract information about transmembrane helix relative positions and their tilt. This information, together with a reliable algorithm to assess the residues involved in each of the membrane spanning regions, can be used to construct a 3D model of the transmembrane domains of rhodopsin at atomic resolution. In the present work, we describe an automated procedure applicable to generate such a model and, in general, to construct a 3D model of any given GPCR with the only assumption that it adopts the same helix arrangement as in rhodopsin. The present approach avoids uncertainties associated with other procedures available for constructing models of GPCRs based on a template, since sequence identity among GPCRs of different families in most of the cases is not significant. The steps involved in the construction of the model are: (i) locate the centers of the helices according to the low-resolution electron density map; (ii) compute the tilt of each helix based on the elliptical shape observed by each helix in the map; (iii) define a local coordinate system for each of the helices; (iv) bring them together in an antiparallel orientation; (v) rotate each helix through the helical axis in such a way that its hydrophobic moment points in the same direction of the bisector formed between three consecutive helices in the bundle; (vi) rotate each helix through an axis perpendicular to the helical one to assign a proper tilt; and (vii) translate each helix to its center deduced from the projection map.     

Comparison of stability predictions and simulated unfolding of rhodopsin structures

Developing a better mechanistic understanding of membrane protein folding is urgently needed because of the discovery of an increasing number of human diseases, where membrane protein instability and misfolding is involved. Towards this goal, we investigated folding and stability of 7-transmembrane (TM) helical bundles by computational methods. We compared the results of three different algorithms for predicting changes in stability of proteins against an experimental mutation dataset obtained for bacteriorhodopsin (BR) and mammalian rhodopsin and find that 61.6% and 70.6% of the mutation results can potentially be explained by known local contributors to the stability of the folded state of BR and mammalian rhodopsin, respectively. To obtain further information on the predicted folding pathway of 7-TM proteins, we conducted simulated thermal unfolding experiments of all available rhodopsin structures with resolution better than 3 angstroms using the Floppy Inclusions and Rigid Substructure Topography (FIRST) method (Jacobs, D. J., A. J. Rader, L. A. Kuhn and M. F. Thorpe [2001] Proteins 44, 150) described previously for a single mammalian rhodopsin structure (Rader et al. [2004] PNAS 101, 7246). In statistical comparison we found that structures of mammalian rhodopsin have a stability core that is characterized by long-range interactions involving amino acids close in space but distant in sequence comprising positions from both extracellular loop and TM regions. In contrast, BR-simulated unfolding does not reveal such a core but is dominated by interactions within individual and groups of TM helices, consistent with the two-stage hypothesis of membrane protein folding. Similar results were obtained for halo- and sensory rhodopsins as for BRs. However, the average folding core energies of sensory rhodopsins were in between those observed for mammalian rhodopsins and BRs hinting at a possible evolution of these structures toward a rhodopsin-like behavior. These results support the conclusion that although the two-stage model can explain the mechanisms of folding and stability of BR, it fails to account for the folding and stability of mammalian rhodopsin, even though the two proteins are structurally related.     

Effect of electrolytes on the cloud point of chlorpromazine hydrochloride solutions

Cloud point (CP) phenomenon occurring in amphiphilic drug chlorpromazine hydrochloride (CPZ) solutions with and without salts is reported herein. The CP of a 50mM CPZ solution (prepared in 10mM sodium phosphate, SP, buffer) was found to decrease with increasing pH, both in the absence as well as presence (50mM) of added salts (NaCl, NaBr, LiBr, KBr, tetra-n-butylammonium bromide). Whereas, at a fixed concentration of NaCl, the CP increased with increasing CPZ concentration, addition of increasing amounts of salts (NaF, NaCl, NaBr, LiCl, KCl) to 50mM CPZ solution (at pH 6.7) caused continuous increase in CP. On the basis of these studies the binding-effect orders of counterions and co-ions have been deduced, respectively, as: Br(-)>Cl(-)>F(-) and Li(+)>Na(+)>K(+). The similar trend of increasing CP with addition of increasing amounts of quaternary bromides (tetramethylammonium bromide, TMeAB; tetraethylammonium bromide, TEtAB; tetra-n-propylammonium bromide, TPrAB; tetra-n-butylammonium bromide, TBuAB; tetra-n-pentylammonium bromide, TPeAB) to 50mM CPZ solutions (at pH 6.7) was found to be dependent upon the alkyl chain length of the particular salt. The overall behaviour has been discussed in terms of electrostatic interactions, micellar growth, and mixed micelle formation.     

Modulation of photosystem II chlorophyll fluorescence by electrogenic events generated by photosystem I.

In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.     

A general procedure for building the transmembrane domains of G‐protein coupled receptors

The only results available at present about the structural features of G‐protein coupled receptors are the low resolution electron projection maps obtained from microscopy studies carried out on two‐dimensional crystals of rhodopsin. These studies support previous suggestions that these integral proteins are constituted by seven transmembrane domains. The low resolution electron density map of rhodopsin can be used to extract information about helix relative positions and tilt. This information, together with a reliable procedure to assess the residues involved in each of the transmembrane regions, can be used to construct a model of rhodopsin at atomic resolution. We have developed an algorithm that can be used to generate such a model in a completely automated fashion. The steps involved are: (i) locate the centers of the helices according to the low resolution electron density map; (ii) compute the tilt of each helix based on the elliptical shape observed by each helix in the map; (iii) define a local coordinate system for each of the helices; (iv) bring them together in an antiparallel orientation; (v) rotate each helix through the helical axis in such a way that its hydrophobic moment points in the same direction as the bisector formed between three consecutive helices in the bundle; (vi) rotate each helix through an axis perpendicular to the helical one to assign a proper tilt; (vii) translate each of the helix to its center deduced from the projection map. A major advantage of the procedure presented is its generality and consequently can be used to obtain a model of any G‐protein coupled receptor with the only assumption that the shape of the bundle is the same as found in rhodopsin. This avoids uncertainties found in other procedures that construct models of G‐protein coupled receptors based on sequence homology using rhodopsin as template. This revised version was published online in July 2006 with corrections to the Cover Date.