Methylation of the active-site lysine of rhodopsin

Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 11-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated at 4 degrees C. Methylation under these conditions leads to the incorporations of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of epsilon-dimethyllysine:epsilon-monomethyllysine:lysine being approximately 5:4:1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a lambda max = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site epsilon-lysine monomethylated rhodopsin with a lambda max = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

His166 is critical for active-site proton transfer and phototaxis signaling by sensory rhodopsin I.

Photoinduced deprotonation of the retinylidene Schiff base in the sensory rhodopsin I transducer (SRI-Htrl) complex results in formation of the phototaxis signaling state S373. Here we report identification of a residue, His166, critical to this process, as well as to reprotonation of the Schiff base during the recovery phase of the SRI photocycle. Each of the residue substitutions A, D, G, L, S, V, or Y at position 166 reduces the flash yield of S373, to values ranging from 2% of wild type for H166Y to 23% for H166V. The yield of S373 is restored to wild-type levels in Htrl-free H166L by alkaline deprotonation of Asp76, a Schiff base proton acceptor normally not ionized in the SRI-Htrl complex, showing that proton transfer from the Schiff base in H166L occurs when an acceptor is made available. The flash yield and rate of decay of S373 of the mutants are pH dependent, even when complexed with Htrl, which confers pH insensitivity to wild-type SRI, suggesting that partial disruption of the complex has occurred. The rates of S373 reprotonation at neutral pH are also prolonged in all H166X mutants, with half-times from 5 s to 160 s (wild type, 1 s). All mutations of His166 tested disrupt phototaxis signaling. No response (H166D, H166L), dramatically reduced responses (H166V), or inverted responses to orange light (H166A, H166G, H166S, and H166Y) or to both orange and near-UV light (H166Y) are observed. Our conclusions are that His166 1) plays a role in the pathways of proton transfer both to and from the Schiff base in the SRI-Htrl complex, either as a structurally important residue or possibly as a participant in proton transfers; 2) is involved in the modulation of SRI photoreaction kinetics by Htrl; and 3) is important in phototaxis signaling. Consistent with the involvement of the His imidazole moiety, the addition of 10 mM imidazole to membrane suspensions containing H166A receptors accelerates S373 decay 10-fold at neutral pH, and a negligible effect is seen on wild-type SRI.     

The molar extinction of rhodopsin

The molar extinction of rhodopsin is 40,600 cm.² per mole equivalent of retinene; i.e., this is the extinction of a solution of rhodopsin which is produced by, or yields on bleaching, a molar solution of retinene. The molar extinctions of all-trans retinene and all-trans retinene oxime have also been determined in ethyl alcohol and aqueous digitonin solutions. On the assumption that each chromophoric group of rhodopsin is made from a single molecule of retinene, it is concluded that the primary photochemical conversion of rhodopsin to lumi-rhodopsin has a quantum efficiency of 1; though the over-all bleaching of rhodopsin in solution to retinene and opsin may have a quantum efficiency as low as one-half. On bleaching cattle rhodopsin, about two sulfhydryl groups appear for each molecule of retinene liberated. In frog rhodopsin the —SH:retinene ratio appears to be higher, 5:2 or perhaps even 3:1. Some of this sulfhydryl appears to have been engaged in binding retinene to opsin; some may have been exposed as the result of changes in opsin which accompany bleaching, comparable with protein denaturation.     

The signaling pathway of rhodopsin

The signal-transduction mechanism of rhodopsin was studied by molecular dynamics (MD) simulations of the high-resolution, inactive structure in an explicit membrane environment. The simulations were employed to calculate equal-time correlations of the fluctuating interaction energy of residue pairs. The resulting interaction-correlation matrix was used to determine a network that couples retinal to the cytoplasmic interface, where transducin binds. Two highly conserved motifs, D(E)RY and NPxxY, were found to have strong interaction correlation with retinal. MD simulations with restraints on each transmembrane helix indicated that the major signal-transduction pathway involves the interdigitating side chains of helices VI and VII. The functional roles of specific residues were elucidated by the calculated effect of retinal isomerization from 11-cis to all-trans on the residue-residue interaction pattern. It is suggested that Glu134 may act as a "signal amplifier" and that Asp83 may introduce a threshold to prevent background noise from activating rhodopsin.     

The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure

A new high-resolution structure is reported for bovine rhodopsin, the visual pigment in rod photoreceptor cells. Substantial improvement of the resolution limit to 2.2 A has been achieved by new crystallization conditions, which also reduce significantly the probability of merohedral twinning in the crystals. The new structure completely resolves the polypeptide chain and provides further details of the chromophore binding site including the configuration about the C6-C7 single bond of the 11-cis-retinal Schiff base. Based on both an earlier structure and the new improved model of the protein, a theoretical study of the chromophore geometry has been carried out using combined quantum mechanics/force field molecular dynamics. The consistency between the experimental and calculated chromophore structures is found to be significantly improved for the 2.2 A model, including the angle of the negatively twisted 6-s-cis-bond. Importantly, the new crystal structure refinement reveals significant negative pre-twist of the C11-C12 double bond and this is also supported by the theoretical calculation although the latter converges to a smaller value. Bond alternation along the unsaturated chain is significant, but weaker in the calculated structure than the one obtained from the X-ray data. Other differences between the experimental and theoretical structures in the chromophore binding site are discussed with respect to the unique spectral properties and excited state reactivity of the chromophore.     

The chaperone environment at the cytoplasmic face of the endoplasmic reticulum can modulate rhodopsin processing and inclusion formation

The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b are type II DnaJ proteins with different C termini generated by alternate splicing. Both protein isoforms can regulate the ATPase activity and substrate binding of Hsp70. In this study, we have confirmed the neuronal expression of HSJ1a and HSJ1b proteins and localized their expression in human neural retina using isoform-specific antisera. HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a geranylgeranyl moiety. Because of their enrichment at the site of rhodopsin production, we investigated the effect of HSJ1 isoforms on the cellular processing of wild-type and mutant rhodopsin apoprotein in SK-N-SH cells. The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild-type rhodopsin-GFP, which co-localized with HSJ1b at the ER. HSJ1b expression also increased the incidence of inclusion formation by the wild-type protein. Both isoforms were recruited to mutant P23H rhodopsin inclusions, but only HSJ1b enhanced inclusion formation. Investigation of a prenylation-null mutant showed that the modulation of rhodopsin processing and inclusion formation was dependent on the correct subcellular targeting of HSJ1b to the cytosolic face of the ER. An Hsp70 interaction-null mutant of HSJ1b had the same effect as HSJ1b, suggesting that these phenomena were independent of Hsp70 and, furthermore, overexpression of Hsp70 with HSJ1b did not modulate the HSJ1b effect on inclusion formation, showing that Hsp70 was not limiting. The data provide evidence that cytoplasmic chaperones when targeted to the ER can influence the folding and processing of a GPCR and show that DnaJ protein function can be further specialized by alternative splicing and post-translational modification.     

The role of active-site residues in naphthalene dioxygenase

The three-component naphthalene dioxygenase enzyme system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. A member of a large family of bacterial Rieske non-heme iron oxygenases, naphthalene dioxygenase is known to oxidize over 60 different aromatic compounds, and many of the products are enantiomerically pure. The crystal structure of the oxygenase component revealed the enzyme to be an α₃β₃ hexamer and identified the amino acids located near the active site. Site-directed mutagenesis studies have identified the residues involved in electron transfer and those responsible for controlling the regioselectivity and enantioselectivity of the enzyme. The results of these studies suggest that naphthalene dioxygenase can be engineered to catalyze a new and extended range of useful reactions.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

The action of enzymes on rhodopsin

The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties.     

Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.     

The enzymes of the transsulfuration pathways: active-site characterizations

The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5'-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine β-lyase) and reverse transsulfuration (cystathionine β-synthase and cystathionine γ-lyase) pathways interconvert l-cysteine and l-homocysteine, the immediate precursor of l-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

The active-site metal coordination geometry of cadmium-substituted alcohol dehydrogenase

 The structure of eleven complexes of cadmium-substituted alcohol dehydrogenase with or without coenzyme and with different non-protein cadmium ligands has been estimated by combined quantum chemical and molecular mechanical geometry optimisations. The geometry of the optimised complexes is similar to the crystal structure of cadmium-substituted alcohol dehydrogenase, indicating that the method behaves well. The optimised structures do not differ significantly (except for the metal bond lengths) from those of the corresponding zinc complexes, which shows that cadmium is a good probe of zinc coordination geometries. The electric field gradients at the cadmium nucleus have been calculated quantum chemically at the MP2 level with a large cadmium basis set, and they have been used to interpret experimental data obtained by perturbed angular correlation of γ-rays. The experimental and calculated field gradients (all three eigenvalues) differ by less than 0.35 a.u. (3.4·10²¹ Vm^–2), the average error is 0.11 a.u., and the average relative error in the two largest eigenvalues of the field gradients is 9%. Calculated field gradients of four-coordinate structures agree better with the experimental results than do those of any five-coordinate model. Thus, the results indicate that the catalytic metal ion remains four-coordinate in all examined complexes. Two measurements are best explained by a four-coordinate cadmium ion with Glu-68 as the fourth ligand, indicating that Glu-68 probably coordinates intermittently to the catalytic metal ion in horse liver alcohol dehydrogenase under physiological conditions. Received: 10 January 1997 / Accepted: 24 May 1997     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

The thermal stability of rhodopsin and opsin

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E_a) and entropies of activation (ΔS‡) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species —frog, squid, cattle—presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.     

Acid-base equilibria in rhodopsin: dependence of the protonation state of glu134 on its environment

Glutamic acid E134 in rhodopsin is part of a highly conserved triad, D(E)RY, located near the cytoplasmic lipid/water interface in transmembrane helix 3 of G protein-coupled receptors (GPCRs). A large body of experimental evidence suggests that the protonation of E134 plays a role in the mechanism of activation of rhodopsin and other GPCRs as well. For E134 to change its protonation state, its pK(a) value must shift from values below physiological pH to higher values. Because of the proximity of the triad to the lipid/water interface, it was hypothesized that a change in solvent around E134 from water to lipid could induce such a shift in pK(a). To test this hypothesis, the pK(a) values of the titratable amino acid residues in rhodopsin have been calculated and the change in solvent around E134 was modeled by shifting the position of the lipid/water interface. The approach used to carry out the pK(a) calculations takes into account the partial immersion of transmembrane proteins in lipid. Qualitative experimental evidence is available for several residues regarding their likely protonation state in rhodopsin at or near physiological pH. Comparison of the calculated pK(a) values with these experimental findings shows good agreement between the two. Notably, glutamic acids E122 and E181 were found to be protonated. The pK(a) values were then calculated for a range of lipid/water interface positions. Although the surrounding solvent of several titratable residues changed from water to lipid in this range, leading to pK(a) shifts in most cases, only for E134 would the shift lead to a change in protonation state at physiological pH. Thus, our results show that the protonation state of E134 is particularly sensitive to its environment. This sensitivity together with the location of E134 near the actual position of the lipid/water interface could be a strategic element in the mechanism of activation of rhodopsin.     

Purification method of bovine rhodopsin kinase using regeneration of rhodopsin

We report a rapid and high-yield purification method of bovine retinal rhodopsin kinase. According to our method, 500 micrograms of rhodopsin kinase was purified from 100 bovine retinae within 12 h. Rhodopsin kinase bound to bleached rhodopsin was extracted effectively from rod outer segment membranes after regeneration of rhodopsin by the incubation with exogenous 11-cis-retinal. Subsequent DE52 column chromatography further purified the protein to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified rhodopsin kinase had an apparent molecular weight of 68,000 and phosphorylated rhodopsin at the rate of 10 nmol phosphate/min/mg of the enzyme.