RESUMEN
In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.
Asunto(s)
Bombas de Protones , Rodopsina , Adenosina Trifosfato/genética , Carbono/metabolismo , Luz , Optogenética , Bombas de Protones/química , Bombas de Protones/genética , Bombas de Protones/metabolismo , Protones , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismo , Rodopsinas Microbianas/genéticaRESUMEN
The functional mechanism of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13-cis retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond. The retinal converts back into an all-trans conformation in the O-intermediate, which is the key state for sodium transport. However, retinal carbon and Schiff base nitrogen chemical shifts differ from those observed in the KR2 dark state all-trans conformation, indicating a perturbation through the nearby bound sodium ion. Our findings are supplemented by optical and infrared spectroscopy and are discussed in the context of known three-dimensional structures.
Asunto(s)
Rodopsina , ATPasa Intercambiadora de Sodio-Potasio , Carbono/metabolismo , Flavobacteriaceae , Iones/metabolismo , Espectroscopía de Resonancia Magnética , Rodopsina/química , Sodio/química , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber's congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber's congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases.
Asunto(s)
Evaluación Preclínica de Medicamentos , Pliegue de Proteína , Rodopsina/química , Rodopsina/metabolismo , Unión Competitiva , Modelos Moleculares , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Rhodopsin, composed of opsin and isomeric retinal, acts as the primary photoreceptor by converting light into electric signals. Inspired by rhodopsin, we have fabricated a light-regulated ionic gate on the basis of the design of a graphene oxide (GO)-biomimetic DNA-nanochannel architecture. In this design, photoswitchable azobenzene (Azo)-DNA is introduced to the surface of porous anodic alumina (PAA) membrane. With modulation of the interaction between the GO blocker and Azo-DNA via flexibly regulating trans and cis states of Azo under the irradiation of visible and ultraviolet light, alternatively, the ionic gate is switched between ON and OFF states. This newly constructed ionic gate can possess high efficiency for the control of ion transport because of the high blocking property of GO and the rather tiny path within the barrier layer which are both first employed to fabricate ionic gate. We anticipate that this rhodopsin-like ionic gate may provide a new model and method for the investigation of ion channel, ion function, and ion quantity. In addition, because of the advantages of simple fabrication, good biocompatibility, and universality, this bioinspired system may have potential applications as optical sensors, in photoelectric transformation, and in controllable drug delivery.
Asunto(s)
Materiales Biomiméticos/química , ADN/química , Grafito/química , Transporte Iónico/efectos de los fármacos , Óxido de Aluminio/química , Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Materiales Biomiméticos/efectos de la radiación , ADN/efectos de la radiación , Técnicas Electroquímicas , Grafito/efectos de la radiación , Transporte Iónico/efectos de la radiación , Membranas Artificiales , Rodopsina/química , Estereoisomerismo , Rayos UltravioletaRESUMEN
Carotenoids are natural pigments with antioxidative functions that protect against oxidative stress. They are essential for humans and must be supplied through the diet. Carotenoids are the precursors for the visual pigment rhodopsin, and lutein and zeaxanthin must be accumulated in the yellow eye spot to protect the retina from excess light and ultraviolet damage. There is a global market for carotenoids as food colorants, animal feed, and nutraceuticals. Some carotenoids are chemically synthesized, whereas others are from natural sources. Microbial mass production systems of industrial interest for carotenoids are in use, and new ones are being developed by metabolic pathway engineering of bacteria, fungi, and plants. Several examples will be highlighted in this chapter.
Asunto(s)
Biotecnología/métodos , Carotenoides/química , Ingeniería Metabólica/métodos , Bacterias/metabolismo , Colorantes/química , Suplementos Dietéticos , Hongos/metabolismo , Microbiología Industrial/métodos , Luz , Luteína/química , Estrés Oxidativo , Plantas/metabolismo , Rodopsina/química , Xantófilas/química , Zeaxantinas/química , beta Caroteno/químicaRESUMEN
An artificial bio-capacitor system is established, consisting of the proton-pump protein proteorhodopsin and a modified alumina nanochannel, inspired by the capacitor-like behavior of plasma membranes realized through the cooperation of ion-pump and ion-channel proteins. Capacitor-like features of this simplified system are realized and identified, and the photocurrent duration time can be modulated by nanochannel modification to obtain favorable square-wave currents.
Asunto(s)
Óxido de Aluminio/química , Materiales Biomiméticos/química , Equipos y Suministros Eléctricos , Luz , Nanotecnología/métodos , Rodopsina/química , Diseño de Equipo , Ensayo de Materiales , Membranas Artificiales , Rodopsinas MicrobianasRESUMEN
The biophysical properties of the lipid matrix are known to influence function of integral membrane proteins. We report on a sample preparation method for reconstitution of membrane proteins which uses porous anodic aluminum oxide (AAO) filters with 200-nm-wide pores of high density. The substrate permits formation of tubular, single membranes that line the inner surface of pores. One square centimeter of filter with a thickness of 60µm yields on the order of 500cm(2) of solid-supported single bilayer surface, sufficient for NMR studies. The tubular bilayers are free of detergent, fully hydrated, and accessible for ligands from one side of the membrane. The use of AAO filters greatly improves reproducibility of the reconstitution process such that the influence of protein on lipid order parameters can be studied with high resolution. As an example, results for the G protein-coupled receptor of class A, bovine rhodopsin, are shown. By (2)H NMR order parameter measurements, it is detected that rhodopsin insertion elastically deforms membranes near the protein. Furthermore, by (1)H saturation-transfer NMR under conditions of magic angle spinning, we demonstrate detection of preferences in interactions of rhodopsin with particular lipid species. It is assumed that function of integral membrane proteins depends on both protein-induced elastic deformations of the lipid matrix and preferences for interaction of the protein with particular lipid species in the first layer of lipids surrounding the protein.
Asunto(s)
Membrana Dobles de Lípidos/química , Resonancia Magnética Nuclear Biomolecular/métodos , Rodopsina/química , Óxido de Aluminio/química , Animales , Bovinos , Ácidos Docosahexaenoicos/química , Elasticidad , Filtración , Fosfatidiletanolaminas/química , Fosfatidilinositoles/química , Fosfatidilserinas/química , Porosidad , Reproducibilidad de los ResultadosRESUMEN
The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.
Asunto(s)
Curcumina/farmacología , Curcumina/uso terapéutico , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/metabolismo , Rodopsina/metabolismo , Animales , Células COS , Chlorocebus aethiops , Inmunohistoquímica , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Retina/efectos de los fármacos , Retina/metabolismo , Retina/patología , Rodopsina/química , Rodopsina/genéticaRESUMEN
In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G betagamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/química , Tirotropina/química , Alanina/química , Animales , Células COS , Dominio Catalítico , Separación Celular , Chlorocebus aethiops , Biología Computacional/métodos , AMP Cíclico/metabolismo , Humanos , Fosfatos de Inositol/química , Modelos Moleculares , Mutación , Estructura Terciaria de ProteínaRESUMEN
The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.
Asunto(s)
Microscopía Confocal/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Animales , Diseño de Equipo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Ratones Transgénicos , Microscopía Confocal/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuronas , Fibras Ópticas , Polen/ultraestructura , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Retina/citología , Retina/metabolismo , Rodopsina/biosíntesis , Rodopsina/química , Rodopsina/genéticaRESUMEN
We report on a novel reconstitution method for G-protein-coupled receptors (GPCRs) that yields detergent-free, single, tubular membranes in porous anodic aluminum oxide (AAO) filters at concentrations sufficient for structural studies by solid-state NMR. The tubular membranes line the inner surface of pores that traverse the filters, permitting easy removal of detergents during sample preparation as well as delivery of ligands for functional studies. Reconstitution of bovine rhodopsin into AAO filters did not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTPgammaS binding assay. The lipid-rhodopsin interaction was investigated by 2H NMR on sn-1- or sn-2-chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as a matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed to spectral density of motions with correlation times in the range of nano- to microseconds, detected as a significant reduction in spin-spin relaxation times. The change in lipid chain order parameters due to interaction with rhodopsin was insignificant.
Asunto(s)
Membrana Dobles de Lípidos/química , Nanopartículas/química , Rodopsina/química , Óxido de Aluminio/química , Óxido de Aluminio/metabolismo , Animales , Bovinos , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/fisiología , Filtración/instrumentación , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ligandos , Luz , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Micelas , Fosfatidilcolinas/química , Porosidad , Unión Proteica/genética , Proteolípidos/química , Proteolípidos/genética , Proteolípidos/metabolismo , Rodopsina/genética , Rodopsina/fisiología , Dispersión de Radiación , Espectrofotometría UltravioletaRESUMEN
G-protein coupled receptors (GPCRs) comprise a large superfamily of proteins that are targets for nearly 50% of drugs in clinical use today. In the past, the use of structure-based drug design strategies to develop better drug candidates has been severely hampered due to the absence of the receptor's three-dimensional structure. However, with recent advances in molecular modeling techniques and better computing power, atomic level details of these receptors can be derived from computationally derived molecular models. Using information from these models coupled with experimental evidence, it has become feasible to build receptor pharmacophores. In this study, we demonstrate the use of the Hybrid Structure Based (HSB) method that can be used effectively to screen and identify prospective ligands that bind to GPCRs. Essentially; this multi-step method combines ligand-based methods for building enriched libraries of small molecules and structure-based methods for screening molecules against the GPCR target. The HSB method was validated to identify retinal and its analogues from a random dataset of approximately 300,000 molecules. The results from this study showed that the 9 top-ranking molecules are indeed analogues of retinal. The method was also tested to identify analogues of dopamine binding to the dopamine D2 receptor. Six of the ten top-ranking molecules are known analogues of dopamine including a prodrug, while the other thirty-four molecules are currently being tested for their activity against all dopamine receptors. The results from both these test cases have proved that the HSB method provides a realistic solution to bridge the gap between the ever-increasing demand for new drugs to treat psychiatric disorders and the lack of efficient screening methods for GPCRs.
Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Diseño Asistido por Computadora , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Rodopsina/química , Rodopsina/genética , Rodopsina/metabolismo , Homología de Secuencia de Aminoácido , Diseño de SoftwareRESUMEN
The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.
Asunto(s)
Receptores de HL/química , Receptores de HL/fisiología , Secuencias de Aminoácidos , Arginina/química , Asparagina/química , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Gonadotropina Coriónica/química , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Citosol/metabolismo , ADN Complementario/metabolismo , Heterocigoto , Humanos , Enlace de Hidrógeno , Leucina/química , Modelos Moleculares , Mutagénesis , Mutación , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/química , Rodopsina/química , Sales (Química)/química , Sales (Química)/farmacología , Programas InformáticosRESUMEN
The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH-/-) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH-/- mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.
Asunto(s)
Oxidorreductasas de Alcohol/fisiología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retinoides/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Animales , Southern Blotting , Catálisis , Línea Celular , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Electrorretinografía , Ojo/metabolismo , Vectores Genéticos , Genotipo , Humanos , Immunoblotting , Inmunohistoquímica , Insectos , Cinética , Luz , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Modelos Químicos , Modelos Genéticos , Mutación , Fosfatidiletanolaminas/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Recombinación Genética , Retinaldehído/química , Retinoides/química , Rodopsina/química , Rodopsina/metabolismo , Factores de Tiempo , Transgenes , Vitamina A/metabolismoRESUMEN
A combined target-based and ligand-based drug design approach has been carried out to define a novel pharmacophore model of the human A(3) receptor antagonists. High throughput molecular docking and comparative molecular field analysis (CoMFA) have been used in tandem to assemble a new target based pharmacophore model. In parallel, to provide more accurate information about the putative binding site of these A(3) inhibitors, a rhodopsin-based model of the human A(3) receptor was built and a novel Y-shape binding motif has been proposed. Docking-based structure superimposition has been used to perform a quantitative study of the structure-activity relationships for binding of these pyrazolo-triazolo-pyrimidines to adenosine A(3) receptor using CoMFA. Both steric and the electrostatic contour plots obtained from the CoMFA analysis nicely fit on the hypothetical binding site obtained by molecular docking. On the basis of the combined hypothesis, we have designed, synthesized, and tested 17 new derivatives. Consistently, the predicted K(i) values were very close to the experimental values.
Asunto(s)
Antagonistas del Receptor de Adenosina A3 , Modelos Moleculares , Pirimidinas/química , Relación Estructura-Actividad Cuantitativa , Triazoles/química , Animales , Sitios de Unión , Técnicas Químicas Combinatorias , Cricetinae , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Modelos Teóricos , Conformación Proteica , Receptor de Adenosina A3/química , Receptor de Adenosina A3/metabolismo , Reproducibilidad de los Resultados , Rodopsina/química , Rodopsina/metabolismo , Electricidad EstáticaRESUMEN
Sterical constraints of the 9-methyl-binding pocket of the rhodopsin chromophore are probed using retinal analogues carrying substituents of increasing size at the 9 position (H, F, Cl, Br, CH(3), and I). The corresponding 11-Z retinals were employed to investigate formation of photosensitive pigment, and the primary photoproduct was identified by Fourier transform infrared difference spectroscopy. In addition, any effects of cumulative strain were studied by introduction of the 9-Z configuration and/or the alpha-retinal ring structure. Our results show that the 9-F analogue still can escape from the 9-methyl-binding pocket and that its photochemistry behaves very similar to the 9-demethyl analogue. The 9-Cl and 9-Br analogues behave very similar to the native 9-methyl pigments, but the 9-I retinal does not fit very well and shows poor pigment formation. This puts an upper limit on the radial dimension of the 9-methyl pocket at 0.45-0.50 nm. Introduction of the alpha-retinal ring constraint in the 11-Z series results in cumulative strain, because the 9-I and 9-Br derivatives cannot bind to generate a photopigment. The 9-Z configuration can partially compensate for the additional alpha-retinal strain. The corresponding 9-Br analogue does form a photopigment, and the other derivatives give increased photopigment yields compared to the corresponding 11-Z derivatives. In fact, 9-Z-alpha-retinal would be an interesting candidate for retinal supplementation studies. Our data provide direct support for the concept that the 9-methyl group is an important determinant in ligand anchoring and activation of the protein and in general agree with a three-point interaction model involving the ring, 9-methyl group, and aldehyde function.
Asunto(s)
Halógenos/metabolismo , Retinaldehído/análogos & derivados , Retinaldehído/síntesis química , Retinaldehído/metabolismo , Rodopsina/análogos & derivados , Rodopsina/síntesis química , Rodopsina/metabolismo , Animales , Sitios de Unión , Bovinos , Congelación , Isomerismo , Ligandos , Unión Proteica , Rodopsina/química , Opsinas de Bastones/química , Opsinas de Bastones/metabolismo , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.
Asunto(s)
Mutación , Receptores de HL/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , AMP Cíclico/metabolismo , Citosol/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Genes Reporteros , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Oligonucleótidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Rodopsina/química , Homología de Secuencia de Aminoácido , Programas Informáticos , Factores de Tiempo , Transfección , Tirosina/químicaRESUMEN
Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6-8-week-old Lrat-/- mice revealed that the rod outer segments are approximately 35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.
Asunto(s)
Aciltransferasas/fisiología , Ésteres/metabolismo , Ojo/metabolismo , Hígado/metabolismo , Aciltransferasas/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión , ADN Complementario/metabolismo , Electrorretinografía , Vectores Genéticos , Genotipo , Homocigoto , Inmunohistoquímica , Cinética , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica , Microscopía Fluorescente , Modelos Genéticos , Fotones , Retina/ultraestructura , Retinoides/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
Retinoid-binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol-binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol-binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand-binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplasmic matrix. All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes. Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.
Asunto(s)
Mariposas Diurnas/metabolismo , Retina/metabolismo , Proteínas de Unión al Retinol/química , Proteínas de Unión al Retinol/metabolismo , Vitamina A/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Citoplasma/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Ligandos , Luz , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión al Retinol/genética , Proteínas Plasmáticas de Unión al Retinol , Rodopsina/química , Espectrometría de Fluorescencia , Espectrofotometría , Temperatura , Factores de Tiempo , Rayos Ultravioleta , Vitamina A/metabolismoRESUMEN
Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.