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1.
Int J Mol Sci ; 24(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36614223

RESUMO

The pathogenic variant p.G90D in RHO is believed to be responsible for a spectrum of phenotypes, including congenital stationary blindness (for the purpose of this study termed night blindness without degeneration; NBWD), Sector RP, Pericentral RP, and Classic RP. We present a correlation between the serum concentration of vitamin A and disease severity in patients with this variant. This prospective study involved 30 patients from 7 families (17 male; median age 46 years, range 8-73). Full ophthalmological examination including visual acuity, Goldmann perimetry, slit-lamp exam, optical coherence tomography, fundus autofluorescence, and electrophysiology was performed to determine the presenting phenotype. The serum concentration of vitamin A was determined from a fasting blood sample taken on the day of the exam, where it was found that 23.3% (7/30) of patients had NBWD, 13.3% (4/30) had Sector RP, 3.3% (1/30) had Pericentral RP, and 60% (18/30) had Classic RP. Multiple logistic regression revealed a significantly higher probability of having a milder phenotype (NBWD or Sector RP) in association with younger age (p < 0.05) and a higher concentration of vitamin A (p < 0.05). We hypothesize that vitamin A in its 11-cis-retinal form plays a role in stabilizing the constitutively active p.G90D rhodopsin and its supplementation could be a potential treatment strategy for p.G90D RHO patients.


Assuntos
Retinite Pigmentosa , Vitamina A , Masculino , Humanos , Estudos Prospectivos , Eletrorretinografia , Retinite Pigmentosa/genética , Retinite Pigmentosa/diagnóstico , Fenótipo , Gravidade do Paciente , Mutação , Rodopsina/genética
2.
J Chem Phys ; 158(1): 015101, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36610954

RESUMO

Determining rates of energy transfer across non-covalent contacts for different states of a protein can provide information about dynamic and associated entropy changes during transitions between states. We investigate the relationship between rates of energy transfer across polar and nonpolar contacts and contact dynamics for the ß2-adrenergic receptor, a rhodopsin-like G-protein coupled receptor, in an antagonist-bound inactive state and agonist-bound active state. From structures sampled during molecular dynamics (MD) simulations, we find the active state to have, on average, a lower packing density, corresponding to generally more flexibility and greater entropy than the inactive state. Energy exchange networks (EENs) are computed for the inactive and active states from the results of the MD simulations. From the EENs, changes in the rates of energy transfer across polar and nonpolar contacts are found for contacts that remain largely intact during activation. Change in dynamics of the contact, and entropy associated with the dynamics, can be estimated from the change in rates of energy transfer across the contacts. Measurement of change in the rates of energy transfer before and after the transition between states thereby provides information about dynamic contributions to activation and allostery.


Assuntos
Simulação de Dinâmica Molecular , Rodopsina , Conformação Proteica , Rodopsina/química , Entropia , Transferência de Energia
3.
J Phys Chem B ; 127(2): 520-527, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36598865

RESUMO

The biological membrane is a complex two-dimensional fluid, in which various molecular interactions regulate the lateral diffusion of membrane-associated molecules. Pinning of membrane proteins or lipids by extra-membrane proteins impedes the diffusion. In addition, coupling between two monolayer leaflets within a phospholipid bilayer via interdigitation plays important roles, though this effect remains elusive. Here, we fabricate a substrate-supported model membrane with patterned bilayer/monolayer regions to explore the influences of interleaflet coupling. A patterned monolayer of polymerized diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), was lithographically generated and used to form patterned lipid bilayers and monolayers. A phospholipid monolayer was formed on top of the polymerized monolayer. The bilayer/monolayer hybrid membrane was continuous and fluid, but lateral diffusion in the monolayer region was significantly retarded, suggesting the influences of interleaflet coupling. We reconstituted photoreceptor rhodopsin (Rh) and G-protein transducin (Gt) as model transmembrane and peripheral proteins. Rh diffused laterally only in the bilayer region, whereas Gt diffused in both bilayer and monolayer regions. The patterned hybrid bilayer/monolayer membrane reproduces the retarded diffusion and confinement of membrane-bound molecules in a controlled manner and provides insight into the physicochemical and functional roles of semipermeable corrals in the cell membrane.


Assuntos
Bicamadas Lipídicas , Fosfolipídeos , Fosfolipídeos/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Rodopsina/metabolismo , Difusão
4.
Methods Enzymol ; 679: 331-342, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36682869

RESUMO

Microbial rhodopsins are photoreceptive membrane proteins found from diverse microorganisms such as archaea, eubacteria, eukaryotes and viruses. Many microbial rhodopsins possess ion-transport activity by light, such as channels and pumps, and ion-transporting rhodopsins are important tools in optogenetics that control animal behavior by light. Historically, molecular mechanism of rhodopsins has been studied by spectroscopic methods for purified proteins. On the other hand, ion-transport function has to be studied by different methods. This chapter introduces two methods of functional assay of ion-transporting rhodopsins by light. One is a patch clamp method using mammalian cells, and another is an ion-transport assay using pH electrode and microbial cells. These functional assay provides fundamental data of ion-transporting rhodopsins, and thus contributes to evaluation for optogenetic tools.


Assuntos
Rodopsina , Rodopsinas Microbianas , Animais , Rodopsina/química , Rodopsina/metabolismo , Transporte de Íons , Rodopsinas Microbianas/química , Archaea/metabolismo , Bactérias/metabolismo , Mamíferos/metabolismo
5.
Proc Natl Acad Sci U S A ; 120(1): e2214276120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36577071

RESUMO

Rhodopsin is the pigment that enables night vision, whereas cone opsins are the pigments responsible for color vision in bright-light conditions. Despite their importance for vision, cone opsins are poorly characterized at the molecular level compared to rhodopsin. Spectra and kinetics of the intermediate states of human green-cone visual pigment (mid-wavelength sensitive, or MWS opsin) were measured and compared with the intermediates and kinetics of bovine rhodopsin. All the major intermediates of the MWS opsin were recorded in the picosecond to millisecond time range. Several intermediates in MWS opsin appear to have characteristics similar to the intermediates of bovine rhodopsin; however, there are some marked differences. One of the most striking differences is in their kinetics, where the kinetics of the MWS opsin intermediates are slower compared to those of the bovine rhodopsin intermediates.


Assuntos
Visão de Cores , Opsinas dos Cones , Humanos , Animais , Bovinos , Rodopsina , Cinética , Temperatura , Opsinas de Bastonetes , Opsinas , Células Fotorreceptoras Retinianas Cones
6.
J Chem Theory Comput ; 19(1): 293-310, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36516450

RESUMO

We present a computational protocol for the fast and automated screening of excited-state hybrid quantum mechanics/molecular mechanics (QM/MM) models of rhodopsins to be used as fluorescent probes based on the automatic rhodopsin modeling protocol (a-ARM). Such "a-ARM fluorescence screening protocol" is implemented through a general Python-based driver, PyARM, that is also proposed here. The implementation and performance of the protocol are benchmarked using different sets of rhodopsin variants whose absorption and, more relevantly, emission spectra have been experimentally measured. We show that, despite important limitations that make unsafe to use it as a black-box tool, the protocol reproduces the observed trends in fluorescence and it is capable of selecting novel potentially fluorescent rhodopsins. We also show that the protocol can be used in mechanistic investigations to discern fluorescence enhancement effects associated with a near degeneracy of the S1/S2 states or, alternatively, with a barrier generated via coupling of the S0/S1 wave functions.


Assuntos
Corantes Fluorescentes , Rodopsina , Modelos Moleculares , Teoria Quântica
7.
Invest Ophthalmol Vis Sci ; 63(13): 2, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36469028

RESUMO

Purpose: Retinitis pigmentosa (RP) is a heterogeneous group of inherited disorders characterized by photoreceptor degeneration. The rhodopsin gene (RHO) is the most frequent cause of autosomal dominant RP (ADRP), yet it remains unclear how RHO mutations cause heterogeneous phenotypes. Energy failure is a main cause of the secondary cone death during RP progression; however, its role in primary rod death induced by ADRP RHO mutants is unknown. Methods: Three RHO missense mutations were chosen from different clinical classes. Wild-type (WT) RHO and its mutants, P23H (class B1), R135L (class A), and G188R (class B2), were overexpressed in 661w cells, a mouse photoreceptor cell line, and their effects on oxidative phosphorylation (OXPHOS) and aerobic glycolysis were compared separately. Results: Here, we report that energy failure is an early event in the cell death caused by overexpression of WT RHO and its mutants. RHO overexpression leads to OXPHOS deficiency, which might be a result of mitochondrial loss. Nonetheless, only in WT RHO and P23H groups, energy stress triggers AMP-activated protein kinase activation and metabolic reprogramming to increase glycolysis. Metabolic reprogramming impairment in R135L and G188R groups might be the reason why energy failure and cell injury are much more severe in those groups. Conclusions: Our results imply that overexpression of RHO missense mutants have distinct impacts on the two energy metabolic pathways, which might be related to their heterogeneous phenotypes.


Assuntos
Degeneração Retiniana , Retinite Pigmentosa , Camundongos , Animais , Rodopsina/genética , Rodopsina/metabolismo , Retinite Pigmentosa/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Mutação , Metabolismo Energético/genética
8.
Commun Biol ; 5(1): 1370, 2022 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-36517671

RESUMO

The adult Drosophila eye is a powerful model system for phototransduction and neurodegeneration research. However, single cell resolution transcriptomic data are lacking for this tissue. We present single cell RNA-seq data on 1-day male and female, 3-day and 7-day old male adult eyes, covering early to mature adult eyes. All major cell types, including photoreceptors, cone and pigment cells in the adult eye were captured and identified. Our data sets identified novel cell type specific marker genes, some of which were validated in vivo. R7 and R8 photoreceptors form clusters that reflect their specific Rhodopsin expression and the specific Rhodopsin expression by each R7 and R8 cluster is the major determinant to their clustering. The transcriptomic data presented in this report will facilitate a deeper mechanistic understanding of the adult fly eye as a model system.


Assuntos
Proteínas de Drosophila , Drosophila , Feminino , Masculino , Animais , Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados , Rodopsina/genética , Rodopsina/metabolismo , Proteínas de Drosophila/metabolismo , Análise de Sequência de RNA
9.
Int J Mol Sci ; 23(23)2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36499010

RESUMO

Vertebrate and fly rhodopsins are prototypical GPCRs that have served for a long time as model systems for understanding GPCR signaling. Although all rhodopsins seem to become phosphorylated at their C-terminal region following activation by light, the role of this phosphorylation is not uniform. Two major functions of rhodopsin phosphorylation have been described: (1) inactivation of the activated rhodopsin either directly or by facilitating binding of arrestins in order to shut down the visual signaling cascade and thus eventually enabling a high-temporal resolution of the visual system. (2) Facilitating endocytosis of activated receptors via arrestin binding that in turn recruits clathrin to the membrane for clathrin-mediated endocytosis. In vertebrate rhodopsins the shutdown of the signaling cascade may be the main function of rhodopsin phosphorylation, as phosphorylation alone already quenches transducin activation and, in addition, strongly enhances arrestin binding. In the Drosophila visual system rhodopsin phosphorylation is not needed for receptor inactivation. Its role here may rather lie in the recruitment of arrestin 1 and subsequent endocytosis of the activated receptor. In this review, we summarize investigations of fly rhodopsin phosphorylation spanning four decades and contextualize them with regard to the most recent insights from vertebrate phosphorylation barcode theory.


Assuntos
Drosophila , Rodopsina , Animais , Rodopsina/metabolismo , Drosophila/metabolismo , Arrestina/metabolismo , Arrestinas/metabolismo , Fosforilação , Clatrina/metabolismo
10.
J Vis ; 22(12): 20, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36445714

RESUMO

Independent spatiotemporal control of the stimulation of the five photoreceptor classes requires a display with as many primary lights to probe their isolated spatial and temporal responses. No such system exists with suitable performance properties. We present a system to construct a five-primary display from commercially available three-primary digital light processing projectors. It optimizes the set of five primary lights required to maximize the achievable contrast of a single photoreceptor class in a silent substitution protocol, including where the background chromaticity is first specified. From these chosen five primaries, we describe a method to convert complex three-primary (RGB) images to five-primary representations with complete specification of the photoreceptor excitations at each pixel. Key to enabling this multiple display system with a single HDMI connection is a novel control protocol implemented in a deterministic field-programmable gate array controller that splits the data into five video streams to allow nearly synchronous presentation of primary image data through multiple displays. Each pixel is controlled over 9.5 bits for each primary over a single frame for measurement of threshold-level vision. In addition to a large contrast gamut, the Maxwellian view offers high retinal illumination to support the investigation of five opsin-based responses to complex spatiotemporal images with a truly silent substitution protocol, while avoiding the confounding effects of uncontrolled photoreceptor excitations as occurs in four-primary systems. The customizable primaries facilitate this display translation to species with different photoreceptor spectral responses, and the optics are designed for integration into microscopes for use as a stimulus generator in physiological experiments.


Assuntos
Opsinas dos Cones , Humanos , Rodopsina , Retina , Iluminação
11.
Int J Mol Sci ; 23(22)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36430370

RESUMO

Arrestins preferentially bind active phosphorylated G protein-coupled receptors (GPCRs). The middle loop, highly conserved in all arrestin subtypes, is localized in the central crest on the GPCR-binding side. Upon receptor binding, it directly interacts with bound GPCR and demonstrates the largest movement of any arrestin element in the structures of the complexes. Comprehensive mutagenesis of the middle loop of rhodopsin-specific arrestin-1 suggests that it primarily serves as a suppressor of binding to non-preferred forms of the receptor. Several mutations in the middle loop increase the binding to unphosphorylated light-activated rhodopsin severalfold, which makes them candidates for improving enhanced phosphorylation-independent arrestins. The data also suggest that enhanced forms of arrestin do not bind GPCRs exactly like the wild-type protein. Thus, the structures of the arrestin-receptor complexes, in all of which different enhanced arrestin mutants and reengineered receptors were used, must be interpreted with caution.


Assuntos
Arrestina , Rodopsina , Arrestina/metabolismo , Rodopsina/metabolismo , Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ligação Proteica
12.
Science ; 378(6619): eabm8797, 2022 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-36378956

RESUMO

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state-dependent antagonism between neocortical somatostatin-expressing (SST+) and vasoactive intestinal peptide-expressing (VIP+) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.


Assuntos
Potenciais de Ação , Hipocampo , Imagem Molecular , Neurônios , Córtex Visual , Animais , Camundongos , Potenciais de Ação/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Interneurônios/fisiologia , Neurônios/classificação , Neurônios/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Imagem Molecular/métodos , Rodopsina/química , Rodopsina/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Córtex Visual/citologia , Córtex Visual/fisiologia , Fluorescência , Medições Luminescentes
13.
Biochemistry ; 61(23): 2698-2708, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36399519

RESUMO

The proton transfer reaction belongs to one of the key triggers for the functional expression of membrane proteins. Rod and cone opsins are light-sensitive G-protein-coupled receptors (GPCRs) that undergo the cis-trans isomerization of the retinal chromophore in response to light. The isomerization event initiates a conformational change in the opsin protein moiety, which propagates the downstream effector signaling. The final step of receptor activation is the deprotonation of the retinal Schiff base, a proton transfer reaction which has been believed to be identical among the cone opsins. Here, we report an unexpected proton transfer reaction occurring in the early photoreaction process of primate blue-sensitive pigment (MB). By using low-temperature UV-visible spectroscopy, we found that the Lumi intermediate of MB formed in transition from the BL intermediate shows an absorption maximum in the UV region, indicating the deprotonation of the retinal Schiff base. Comparison of the light-induced difference FTIR spectra of Batho, BL, and Lumi showed significant α-helical backbone C=O stretching and protonated carboxylate C=O stretching vibrations only in the Lumi intermediate. The transition from BL to Lumi thus involves dramatic changes in protein environment with a proton transfer reaction between the Schiff base and the counterion resulting in an absorption maximum in the UV region.


Assuntos
Opsinas dos Cones , Pigmentos da Retina , Animais , Pigmentos da Retina/química , Prótons , Bases de Schiff/química , Primatas/metabolismo , Retinaldeído/química , Rodopsina/química
14.
Nat Commun ; 13(1): 6652, 2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36333283

RESUMO

The understanding of how the rhodopsin sequence can be modified to exactly modulate the spectroscopic properties of its retinal chromophore, is a prerequisite for the rational design of more effective optogenetic tools. One key problem is that of establishing the rules to be satisfied for achieving highly fluorescent rhodopsins with a near infrared absorption. In the present paper we use multi-configurational quantum chemistry to construct a computer model of a recently discovered natural rhodopsin, Neorhodopsin, displaying exactly such properties. We show that the model, that successfully replicates the relevant experimental observables, unveils a geometrical and electronic structure of the chromophore featuring a highly diffuse charge distribution along its conjugated chain. The same model reveals that a charge confinement process occurring along the chromophore excited state isomerization coordinate, is the primary cause of the observed fluorescence enhancement.


Assuntos
Retina , Rodopsina , Rodopsina/genética , Rodopsina/química , Fluorescência
15.
Proc Natl Acad Sci U S A ; 119(45): e2213911119, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36322748

RESUMO

For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11-cis-retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography-mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N-retinylidene-phosphatidylethanolamine (N-ret-PE) adducts with the released all-trans-retinal, and the reduction of all-trans-retinal to all-trans-retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all-trans-retinal. In the absence of NADPH, free all-trans-retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N-ret-PE (∼40% of total all-trans-retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N-ret-PE formation was highly attenuated by NADPH-dependent reduction of all-trans-retinal to all-trans-retinol. Neither N-ret-PE formation nor all-trans-retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all-trans-retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.


Assuntos
Retinaldeído , Rodopsina , Rodopsina/metabolismo , Retinaldeído/química , Vitamina A , Hidrólise , NADP
16.
Dis Model Mech ; 15(11)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36420970

RESUMO

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.


Assuntos
Células Fotorreceptoras , Rodopsina , Animais , Humanos , Células Fotorreceptoras/metabolismo , Rodopsina/metabolismo , Transporte Proteico , Mamíferos/metabolismo
17.
J Phys Chem B ; 126(43): 8680-8688, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36281583

RESUMO

Photoreceptor proteins play a critical role in light utilization for energy conversion and environmental sensing. Rhodopsin is a prototypical photoreceptor protein containing a retinal group that functions as a light-receptive site. It is essential to characterize the structure of the retinal chromophore because the chromophore structure, along with retinal-protein interactions, regulates which wavelengths of light are absorbed. Resonance Raman spectroscopy is a powerful tool to characterize chromophore structures in proteins. The resonance Raman spectra of heliorhodopsins, a recently discovered rhodopsin family, were previously reported to exhibit two intense ethylenic C═C stretching bands never observed for type-1 rhodopsins. Here, we show that the double-band feature in the ethylenic C═C stretching modes is not due to structural inhomogeneity but rather to the retinal polyene chain's linear structure. It contrasts with bent all-trans chromophore in type-1 rhodopsins. The linear structure of the chromophore results from weak atomic contacts between the 13-methyl group and a nearby Trp side chain, which can slow thermal reisomerization in the photocycle. It is possible that the deceleration of reisomerization increases the lifetime of the signaling intermediate for photosensory function.


Assuntos
Rodopsina , Rodopsinas Microbianas , Rodopsina/química , Rodopsinas Microbianas/química , Análise Espectral Raman/métodos , Vibração
18.
Phys Chem Chem Phys ; 24(42): 26223-26231, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36278932

RESUMO

Rhodopsins form a family of photoreceptor proteins which utilize the retinal chromophore for light energy conversion. Upon light absorption the retinal chromophore undergoes a photoisomerization. This reaction involves a non-radiative relaxation through a conical intersection between the singlet excited state and the ground state. In this work we studied the possible involvement of triplet states in the photoisomerization of retinaloids using the extended multistate (XMS) version of CASPT2. To this end, truncated models of three retinaloids were considered: protonated Schiff base, deprotonated Schiff base and the aldehyde form. The optimized geometries of the reactant, the product and the conical intersection were connected by a linear interpolation of internal coordinates to describe the isomerization. The energetic position of the low-lying singlet and triplet states as well as their spin-orbit coupling matrix elements (SOCME) were calculated along the isomerization profile. The SOCME values peaked in vicinity of the conical intersection for all the retinaloids. Furthermore, the magnitude of SOCME is invariant to the number of double bonds in the model. The SOCME for the protonated Schiff base is negligible (1.5 cm-1) which renders the involvement of the triplet state as improbable. However, the largest SOCME value of 30 cm-1 was found for the aldehyde form, followed by 15 cm-1 for the deprotonated Schiff base.


Assuntos
Rodopsina , Bases de Schiff , Bases de Schiff/química , Isomerismo , Rodopsina/química , Aldeídos
19.
ACS Synth Biol ; 11(11): 3805-3816, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36264158

RESUMO

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO2 to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO2 fixation. A light-driven proton pump comprising Gloeobacter rhodopsin (GR) and its cofactor retinal have been assembled in Ralstonia eutropha (Cupriavidus necator) H16. In the presence of light, this strain fixed inorganic carbon (or bicarbonate) leading to 20% growth enhancement, when formate was used as an electron donor. We found that an electrode from a solar panel can replace organic compounds to serve as the electron donor, mediated by the electron shuttle molecule riboflavin. In this new autotrophic and photo-electrosynthetic system, GR is augmented by an external photocell for reductive CO2 fixation. We demonstrated that this hybrid photo-electrosynthetic pathway can drive the engineered R. eutropha strain to grow using CO2 as the sole carbon source. In this system, a bioreactor with only two inputs, light and CO2, enables the R. eutropha strain to perform a rhodopsin-dependent autotrophic growth. Light energy alone, supplied by a solar panel, can drive the conversion of CO2 into biomass with a maximum electron transfer efficiency of 20%.


Assuntos
Cupriavidus necator , Rodopsina , Rodopsina/genética , Rodopsina/metabolismo , Dióxido de Carbono/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Processos Autotróficos , Carbono/metabolismo
20.
Biochemistry (Mosc) ; 87(10): 1187-1198, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36273887

RESUMO

Due to the unique capability of modulating cell membrane potential upon photoactivation, channelrhodopsins of green (Chlorophyta) and cryptophytic (Cryptophyta) algae are widely employed in optogenetics, a modern method of light-dependent regulation of biological processes. To enable the search for new genes perspective for optogenetics, we have developed the PCR tests for the presence of genes of the cation and anion channelrhodopsins. Six isolates of green algae Haematococcus and Bracteacoccus from the White Sea region and 2 specimens of Rhodomonas sp. (Cryptophyta) from the regions of White and Black Seas were analyzed. Using our PCR test we have demonstrated the known Haematococcus rhodopsin genes and have discovered novel rhodopsin genes in the genus of Bracteacoccus. Two distantly homologous genes of anion channelrhodopsins were also identified in the cryptophytic Rhodomonas sp. from the White and Black Seas. These results indicate that the developed PCR tests might be useful tool for a broad-range screening of the Chlorophyta and Cryptophyta algae to identify unique channelrhodopsin genes.


Assuntos
Criptófitas , Rodopsina , Channelrhodopsins/metabolismo , Criptófitas/genética , Criptófitas/metabolismo , Rodopsina/genética , Mar Negro , Optogenética/métodos , Ânions , Cátions
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