RESUMO
Absorption of light in rhodopsin leads through 11-cis- and all-trans-retinal isomerization, proton transfers, and structural changes to the active G-protein binding meta-II state. When meta-II is photolysed by blue light absorption, the activating pathway is apparently reverted, and rhodopsin is photoregenerated. However, the product formed, a P subspecies with A(max) = 500 nm (P(500)), is different from the ground state based on the following observations: (i) the ground state fingerprint of 11-cis-retinal does not appear in the infrared spectra, although the proton transfers and structural changes are reverted; (ii) extraction of the retinal from P(500) does not yield the expected stoichiometric amount of 11-cis-retinal but predominantly yields all-trans-retinal; (iii) the infrared spectrum of P(500) is similar to the classical meta-III intermediate, which arises from meta-II by thermal decay; and (iv) both P(500) and meta-III can be photoconverted to meta-II with the same changes in the infrared spectrum and without a significant change in the isomerization state of the extracted chromophore. The data indicate the presence of a "second switch" between active and inactive conformations that operates by photolysis but without isomerization around the C(11)-C(12) double bond. This emphasizes the exclusivity of the ground state, which is only accessible by the metabolic regeneration with 11-cis-retinal.
Assuntos
Luz , Rodopsina/química , Rodopsina/metabolismo , Cromatografia Líquida de Alta Pressão , Modelos Biológicos , Modelos Químicos , Ligação Proteica , Prótons , Rodopsina/análogos & derivados , Transdução de Sinais , Espectrofotometria , Estereoisomerismo , Fatores de TempoRESUMO
G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.
Assuntos
Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Citoplasma/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Fótons , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Doenças Retinianas/metabolismo , Transdução de SinaisRESUMO
The guanine nucleotide-binding protein (G-protein)-coupled receptor superfamily (GPCR) is comprised of a large group of membrane proteins involved in a wide range of physiological signaling processes. The functional switch from a quiescent to an active conformation is at the heart of GPCR action. The GPCR rhodopsin has been studied extensively because of its key role in scotopic vision. The ground state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Light induces cis-trans isomerization and rhodopsin activation. Here we show that rhodopsin regenerated with a ring-constrained 11-cis-retinal analog undergoes photoisomerization; however, it remains marginally active because isomerization occurs without the chromophore-induced conformational change of the opsin moiety. Modeling the locked chromophore analogs in the active site of rhodopsin suggests that the beta-ionone ring rotates but is largely confined within the binding site of the natural 11-cis-retinal chromophore. This constraint is a result of the geometry of the stable 11-cis-locked configuration of the chromophore analogs. These results suggest that the native chromophore cis-trans isomerization is merely a mechanism for repositioning of the beta-ionone ring which ultimately leads to helix movements and determines receptor activation.
Assuntos
Rodopsina/química , Animais , Bovinos , Conformação Proteica , Retina/metabolismo , Rodopsina/metabolismo , Relação Estrutura-AtividadeRESUMO
The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (<10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.
Assuntos
Fosfolipídeos/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiação , Animais , Transporte Biológico , Bovinos , Luz , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Marcadores de Spin , Transducina/metabolismoRESUMO
Despite the growing structural information on receptors and G proteins, the information on affinities and kinetics of protein-protein and protein-nucleotide interactions is still not complete. In this study on photoactivated rhodopsin (R*) and the rod G protein, G(t), we have used kinetic light scattering, backed by direct biochemical assays, to follow G protein activation. Our protocol includes the following: (i) to measure initial rates on the background of rapid depletion of the G(t)GDP substrate; (ii) to titrate G(t)GDP, GTP, and GDP; and (iii) to apply a double displacement reaction scheme to describe the results. All data are simultaneously fitted by one and the same set of parameters. We obtain values of K(m) = 2200 G(t)/microm(2) for G(t)GDP and K(m) = 230 microm for GTP; dissociation constants are K(d) = 530 G(t)/microm(2) for R*-G(t)GDP dissociation and K(d) = 270 microm for GDP release from R*G(t)GDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 G(t)/s at 22 degrees C and 1300 G(t)/s at 34 degrees C. The analysis provides a tool to allocate and quantify better the effects of chemical or mutational protein modifications to individual steps in signal transduction.
Assuntos
Rodopsina/química , Rodopsina/metabolismo , Transducina/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Ligantes , Luz , Modelos Químicos , Ligação Proteica , Retina/metabolismo , Espalhamento de Radiação , Transdução de Sinais , Temperatura , TermodinâmicaRESUMO
To perform their functions within an organism, or to adapt to the environment as single cells, living cells react to signals detected by highly specialized receptor proteins. These include the G-protein coupled receptors (GPCRs), a receptor family, which comprises more than 1000 members, and is of outstanding significance in basic research and medical application. An archetype of a GPCR is the visual pigment rhodopsin, the photoreceptor of the retinal rod cell. Biophysical methods have largely contributed to the elucidation of rhodopsin structure and function, as well as of the corresponding signal cascade. This article discusses some of the more recent developments.
Assuntos
Luz , Transdução de Sinais/fisiologia , Visão Ocular/fisiologia , Animais , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/química , Rodopsina/fisiologiaRESUMO
Arrestin blocks the interaction of rhodopsin with the G protein transducin (G(t)). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G(t) and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10-30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11-30 and 51-70 (IC(50) < 100 microm) and residues 231-260 (IC(50) < 200 microm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G(t) or the farnesylated G(t)gamma C-terminal peptide. Only arrestin-(11-30) inhibited MII.G(t) less (IC(50) = 300 microm) than phosphorylated MII.arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and G(t). The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure.
Assuntos
Arrestina/metabolismo , Rodopsina/análogos & derivados , Rodopsina/metabolismo , Transducina/metabolismo , Sequência de Aminoácidos , Animais , Arrestina/química , Ligação Competitiva , Bovinos , Luz , Modelos Moleculares , Dados de Sequência Molecular , Rodopsina/química , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiaçãoRESUMO
The oscillating drop surfactometer (ODS) measures surface tension (gamma) and energy dissipation (damping constant b) of surfactant on a 1 microl sample. gamma is obtained from the period of oscillation and b from its free decay or from the phase shift slope in resonance. After calibration with substances with different gamma, corrections were made for capillary fixation and loss of mass by evaporation. Surface active substances are delivered from liposomes in the interior (subphase) or injected from outside, with microdrops (180 pl each) of solution. As an application example, we have investigated surfactant extract and pure phospholipid. In minutes after formation of a drop containing a diluted Survanta suspension, gamma, decreases by 20 mN/m, while b increases three-fold. This effect, assigned to spontaneous adsorption from liposomes to the surface, is not seen with pure dipalmitoylphosphatidylcholine (DPPC) under our conditions. However, microdrop injection of DPPC triggers a rapid decrease of gamma and a delayed strong increase in b. The effect is modulated by DPPC in the subphase and by cholesterol. Investigations with L-alpha-lysophosphatidylcholine show the high sensitivity of the ODS technique in the determination of the energy dissipation at air-liquid boundary surfaces. Although the ODS is limited to applications with gamma > 15 mN m(-1), it offers the advantage to give, with small samples and within seconds, a simultaneous readout of both Surface tension gamma and the parameter b, as a measure of surface viscosity.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Produtos Biológicos , Lipossomos/química , Lisofosfatidilcolinas/química , Surfactantes Pulmonares/química , Animais , Ação Capilar , Bovinos , Colesterol/química , Oscilometria , Tensão Superficial , ViscosidadeRESUMO
Metarhodopsin II (MII) provides the active conformation of rhodopsin for interaction with the G-protein, Gt. Fourier transform infrared spectra from samples prepared by centrifugation reflect the pH dependent equilibrium between MII and inactive metarhodopsin I. C-terminal synthetic peptides (Gtalpha(340-350) and Gtgamma(60-71)farnesyl) stabilize MII. We find that both peptides cause similar spectral changes not seen with control peptides (Gtalpha (K341R, L349A) and non-farnesylated Gtgamma). The spectra reflect all the protonation dependent bands normally observed when MII is formed at acidic pH. Beside the protonation dependent bands, additional features, similar with both peptides, appear in the amide I and II regions.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Rodopsina/análogos & derivados , Espectroscopia de Infravermelho com Transformada de Fourier , Animais , Bovinos , Concentração de Íons de Hidrogênio , Cinética , Prótons , Rodopsina/química , Segmento Externo da Célula Bastonete/químicaRESUMO
The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.
Assuntos
Prótons , Retinaldeído/química , Rodopsina/química , Rodopsina/metabolismo , Transdução de Sinais , Animais , Células COS , Bovinos , Olho/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Concentração de Íons de Hidrogênio , Luz , Modelos Biológicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Retinaldeído/análogos & derivados , Bases de Schiff/química , Análise Espectral , Transducina/química , Raios UltravioletaRESUMO
The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal-protein interaction.
Assuntos
Proteínas do Olho , Retinaldeído/metabolismo , Rodopsina/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Arrestina/isolamento & purificação , Arrestina/metabolismo , Bovinos , Membrana Celular/química , Membrana Celular/metabolismo , Receptor Quinase 1 Acoplada a Proteína G , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Fosforilação , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Retina/metabolismo , Rodopsina/química , Rodopsina/isolamento & purificação , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/metabolismo , Opsinas de Bastonetes/química , Opsinas de Bastonetes/isolamento & purificação , Espectrofotometria/métodos , Transducina/isolamento & purificação , Transducina/metabolismoAssuntos
Arrestina/metabolismo , Proteínas do Olho , Diester Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Transducina/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Receptor Quinase 1 Acoplada a Proteína G , Cinética , Luz , Ligação Proteica , Espalhamento de Radiação , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Visão OcularAssuntos
Concentração de Íons de Hidrogênio , Rodopsina/química , Rodopsina/metabolismo , Calibragem , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Luz , Prótons , Bases de Schiff , Espectrofotometria Ultravioleta/instrumentação , Espectrofotometria Ultravioleta/métodos , Visão OcularAssuntos
Rodopsina/química , Rodopsina/metabolismo , Transducina/química , Transducina/metabolismo , Biofísica/métodos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Luz , Bicamadas Lipídicas , Fotólise , Espalhamento de Radiação , Espectrometria de Fluorescência/métodos , Espectrofotometria/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spin-label. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of < 5 min at 25 degrees C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.
Assuntos
Bicamadas Lipídicas , Fosfolipídeos/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Etilmaleimida/farmacologia , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Marcadores de SpinRESUMO
In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.
Assuntos
Ácido Palmítico/metabolismo , Retinaldeído/metabolismo , Opsinas de Bastonetes/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Fluorescência , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Concentração de Íons de Hidrogênio , Isomerismo , Luz , Dados de Sequência Molecular , Palmitoil Coenzima A/metabolismo , Ligação Proteica , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Espectrofotometria , Transducina/metabolismoRESUMO
The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.
Assuntos
Rodopsina/química , Rodopsina/metabolismo , Transducina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biofísica/métodos , Bovinos , Relação Dose-Resposta a Droga , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Rodopsina/genética , Fatores de TempoRESUMO
Functional coupling of the human thrombin receptor PAR1 (protease-activated receptor 1) with the retinal rod G-protein transducin (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent Gt activation was measured using a real-time fluorimetric GTP[S]-binding assay and membranes from Sf9 cells. To characterize nucleotide-exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC50 = 5.2 +/- 1.5 microM at 5 microM GTP[S]), indicating that receptor-Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 degrees C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 +/- 3.5 kJ.mol-1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa of 6.4. This supports the idea that the active state of agonist-bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.
Assuntos
Receptores de Trombina/metabolismo , Transducina/metabolismo , Animais , Bovinos , Linhagem Celular , Expressão Gênica , Nucleotídeos de Guanina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Receptor PAR-1 , Receptores de Trombina/genética , Rodopsina/metabolismo , SpodopteraRESUMO
In microseconds after photoexcitation, rhodopsin forms the Meta I intermediate from lumirhodopsin. In this conversion, contacts between retinal and the apoprotein are formed, which result in a defined arrangement of donor and acceptor groups for proton translocations. A system of protonation-dependent coupled equilibria is now adopted, comprising Meta intermediates I, II and III, and their isospectral subforms. Some Meta states were identified as signalling states, in which the receptor interacts with transducin (Gt), rhodopsin kinase (RK) and arrestin. The binding of Gt or arrestin shifts the equilibrium to Meta II, while RK does not, indicating exposure of the RK binding site(s) before Meta II is formed. On contact with the activated receptor, each signalling protein responds with a conformational change, which transforms it into a functionally active state. The bell-shaped pH/rate profiles which are seen for the activation of both the G protein and the receptor kinase, indicate the necessary protonation and deprotonation of groups with different pKa. The right wing of the profile reflects the formation of the protonated subconformation (termed MIIb) of Meta II. For the interaction with Gt, recent work suggests a 'sequential fit' mechanism, involving the recognition of the C-terminal peptide of the Gt alpha subunit and of the farnesylated C-terminus of the gamma subunit. Isolated peptides derived from these portions of the G protein mimic the left wing of the pH/rate profile. We discuss the sequential fit as a time-ordered sequence of microscopic recognition and conformational interlocking in the interaction with the G protein.