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1.
Biochemistry ; 60(41): 3050-3057, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34601881

RESUMO

A transmembrane proton gradient is generated and maintained by proton pumps in a cell. Metagenomics studies have recently identified a new category of rhodopsin intermediates between type-1 rhodopsins and heliorhodopsins, named schizorhodopsins (SzRs). SzRs are light-driven inward proton pumps. Comprehensive resonance Raman measurements were conducted to characterize the structure of the retinal chromophore in the unphotolyzed state of four SzRs. The spectra of all four SzRs show that the retinal chromophore is in the all-trans and 15-anti configuration and that the Schiff base is protonated. The polyene chain is planar in the center of the retinal chromophore and is twisted in the vicinity of the protonated Schiff base. The protonated Schiff base in the SzRs forms a stronger hydrogen bond than that in outward proton-pumping rhodopsins. We determined that the hydrogen-bonding partner of the protonated Schiff base is not a water molecule but an amino acid residue, presumably an Asp residue in helix G. The present observations provide valuable insights into the inward proton-pumping mechanism of SzRs.


Assuntos
Proteínas Arqueais/química , Polienos/química , Bombas de Próton/química , Rodopsinas Microbianas/química , Bases de Schiff/química , Archaea/química , Ligação de Hidrogênio
2.
Angew Chem Int Ed Engl ; 60(42): 23010-23017, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34339559

RESUMO

The new class of microbial rhodopsins, called xenorhodopsins (XeRs),[1] extends the versatility of this family by inward H+ pumps.[2-4] These pumps are an alternative optogenetic tool to the light-gated ion channels (e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).[1] The photodynamic behavior of NsXeR was investigated on the ps to s time scale elucidating the formation of the J and K and a previously unknown long-lived intermediate. The pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover. In patch-clamp experiments the blue-light illumination of NsXeR in the M state shows a potential-dependent vectoriality of the photocurrent transients, suggesting a variable accessibility of reprotonation of the retinal Schiff base. Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H+ acceptor D220.


Assuntos
Bombas de Próton/metabolismo , Rodopsinas Microbianas/metabolismo , Bases de Schiff/química , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Cinética , Luz , Optogenética , Bombas de Próton/química , Prótons , Rodopsinas Microbianas/química , Espectrofotometria , Temperatura
3.
J Phys Chem B ; 125(31): 8797-8804, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34342994

RESUMO

Heliorhodopsins are a recently discovered diverse retinal protein family with an inverted topology of the opsin where the retinal protonated Schiff base proton is facing the cell cytoplasmic side in contrast to type 1 rhodopsins. To explore whether light-induced retinal double-bond isomerization is a prerequisite for triggering protein conformational alterations, we utilized the retinal oxime formation reaction and thermal denaturation of a native heliorhodopsin of Thermoplasmatales archaeon SG8-52-1 (TaHeR) as well as a trans-locked retinal analogue (TaHeRL) in which the critical C13═C14 double-bond isomerization is prevented. We found that both reactions are light-accelerated not only in the native but also in the "locked" pigment despite lacking any isomerization. It is suggested that light-induced charge redistribution in the retinal excited state polarizes the protein and triggers protein conformational perturbations that thermally decay in microseconds. The extracted activation energy and the frequency factor for both the reactions reveal that the light enhancement of TaHeR differs distinctly from the earlier studied type 1 microbial rhodopsins.


Assuntos
Rodopsina , Rodopsinas Microbianas , Luz , Conformação Proteica , Retina , Retinaldeído , Bases de Schiff
4.
Molecules ; 26(15)2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-34361639

RESUMO

Many experiments have been carried out to display different colors of Proteorhodopsin (PR) and its mutants, but the mechanism of color tuning of PR was not fully elucidated. In this study, we applied the Electrostatically Embedded Generalized Molecular Fractionation with Conjugate Caps (EE-GMFCC) method to the prediction of excitation energies of PRs. Excitation energies of 10 variants of Blue Proteorhodopsin (BPR-PR105Q) in residue 105GLN were calculated with the EE-GMFCC method at the TD-B3LYP/6-31G* level. The calculated results show good correlation with the experimental values of absorption wavelengths, although the experimental wavelength range among these systems is less than 50 nm. The ensemble-averaged electric fields along the polyene chain of retinal correlated well with EE-GMFCC calculated excitation energies for these 10 PRs, suggesting that electrostatic interactions from nearby residues are responsible for the color tuning. We also utilized the GMFCC method to decompose the excitation energy contribution per residue surrounding the chromophore. Our results show that residues ASP97 and ASP227 have the largest contribution to the absorption spectral shift of PR among the nearby residues of retinal. This work demonstrates that the EE-GMFCC method can be applied to accurately predict the absorption spectral shifts for biomacromolecules.


Assuntos
Simulação de Dinâmica Molecular , Teoria Quântica , Rodopsinas Microbianas/química , Eletricidade Estática
5.
J Photochem Photobiol B ; 223: 112285, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34411952

RESUMO

Microbial pumping rhodopsin is a seven-transmembrane retinal binding protein, which is light-driven ion pump with a functional key motif. Ion-pumping with the key motif and charged amino acids in the rhodopsin is biochemically important. The rhodopsins with DTG motif have been discovered in various eubacteria, and they function as H+ pump. Especially, the DTG motif rhodopsins transported H+ despite the replacement of a proton donor by Gly. We investigated Methylobacterium populi rhodopsin (MpR) in one of the DTG motif rhodopsin clades. To determine which ions the MpR transport, we tested with various monovalent ion solutions and determined that MpR transports Li+/Na+. By replacing the three negatively charged residues residues which are located in helix B, Glu32, Glu33, and Asp35, we concluded that the residues play a critical role in the transport of Li+/Na+. The MpR E33Q transported H+ in place of Li+/Na+, suggesting that Glu33 is a Li+/Na+ binding site on the cytoplasmic side. Gly93 in MpR was replaced by Asp to convert from the Li+/Na+ pump to the H+ pump, resulting in MpR G93D transporting H+. Dissociation constant (Kd) values of Na+ for MpR WT and E33Q were determined to be 4.0 and 72.5 mM, respectively. These results indicated the mechanism by which MpR E33Q transports H+. Up to now, various ion-pumping rhodopsins have been discovered, and Li+/Na+-pumping rhodopsins were only found in the NDQ motif in NaR. Here, we report a new light-driven Na+ pump MpR and have determined the important residues required for Li+/Na+-pumping different from previously known NaR.


Assuntos
Lítio/metabolismo , Rodopsinas Microbianas/metabolismo , Sódio/metabolismo , Motivos de Aminoácidos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos da radiação , Luz , Lítio/química , Methylobacteriaceae/metabolismo , Mutagênese Sítio-Dirigida , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rodopsinas Microbianas/química , Rodopsinas Microbianas/classificação , Rodopsinas Microbianas/genética , Sódio/química
6.
Nat Commun ; 12(1): 4107, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226545

RESUMO

The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.


Assuntos
Microscopia Crioeletrônica , Luz , Rodopsina/química , Rodopsinas Microbianas/química , Expressão Gênica , Simulação de Dinâmica Molecular , Fenômenos Físicos , Conformação Proteica , Prótons
7.
J Phys Chem B ; 125(30): 8331-8341, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34292728

RESUMO

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) α-helices and an all-trans-retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins. The X-ray crystal structure of HeR 48C12 was elucidated after the first report on a HeR variant from Thermoplasmatales archaeon SG8-52-1, which revealed the water-mediated hydrogen-bonding network connected to the Schiff base region in the cytoplasmic side. Herein, low-temperature light-induced FTIR spectroscopic analyses of HeR 48C12 and 15N isotopically labeled proteins were used to elucidate the structural changes during retinal photoisomerization. N-D stretching vibrations of the protonated retinal Schiff base (PRSB) at 2286 and 2302 cm-1 in the dark state, and 2239 and 2252 cm-1 in the K intermediate were observed. The frequency changes indicated that the hydrogen bond of PRSB strengthens upon photoisomerization in HeR. Moreover, O-D stretching vibration frequencies of the internal water molecules indicate that the hydrogen-bonding strength decreases concomitantly. Therefore, the PRSB hydrogen bond responds to photoisomerization in an opposite way to the hydrogen-bonding network involving water molecules. No frequency changes of the indole N-H or N-D stretching vibrations of tryptophan residues were observed upon photoisomerization, suggesting that all tryptophan residues in the HeR 48C12 maintained the hydrogen-bonding strengths in the K intermediate. These results provide insights into the molecular mechanism of the energy storage and propagation upon retinal photoisomerization in HeR.


Assuntos
Bacteriorodopsinas , Bases de Schiff , Hidrogênio , Ligação de Hidrogênio , Rodopsinas Microbianas , Espectroscopia de Infravermelho com Transformada de Fourier , Água
8.
J Photochem Photobiol B ; 221: 112241, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34130090

RESUMO

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.


Assuntos
Cantaxantina/química , Rodopsinas Microbianas/química , Energia Solar , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteroidetes/metabolismo , Sítios de Ligação , Calorimetria , Cantaxantina/metabolismo , Cianobactérias/metabolismo , Luz , Ligação Proteica , Rodopsinas Microbianas/metabolismo , Alinhamento de Sequência
9.
J Phys Chem B ; 125(26): 7155-7162, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34167296

RESUMO

Light is utilized as energy or information by rhodopsins (membrane proteins that contain a retinal chromophore). Heliorhodopsins (HeRs) are a new class of rhodopsins with low sequence identity (<15%) to microbial and animal rhodopsins. Their physiological roles remain unknown, although the involvement of a long-lived intermediate in the photocycle suggests a light-sensor function. Characterization of the molecular structures of the intermediates is essential to an understanding of the roles and mechanisms of HeRs. We determined the chromophore structures of the intermediates in HeR 48C12 by time-resolved resonance Raman spectroscopy and observed that the hydrogen bond of the protonated Schiff base strengthened prior to deprotonation. The chromophore is photoisomerized from the all-trans to the 13-cis form and is reisomerized in the transition from the O intermediate to the unphotolyzed state. Our results demonstrate that the chromophore structure evolves similarly to microbial rhodopsins, despite the dissimilarity in amino acid residues surrounding the chromophore.


Assuntos
Rodopsinas Microbianas , Vibração , Ligação de Hidrogênio , Estrutura Molecular , Rodopsina , Bases de Schiff , Análise Espectral Raman
10.
Proc Natl Acad Sci U S A ; 118(14)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33790007

RESUMO

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the ß-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.


Assuntos
Bombas de Próton/química , Rodopsinas Microbianas/química , Sítios de Ligação , Escherichia coli , Conformação Proteica
11.
Sheng Wu Gong Cheng Xue Bao ; 37(2): 604-614, 2021 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-33645158

RESUMO

Proton-pumping rhodopsin (PPR) is a simple photosystem widely distributed in nature. By binding to retinal, PPR can transfer protons from the cytoplasmic to the extracellular side of the membrane under illumination, creating a proton motive force (PMF) to synthesize ATP. The conversion of light into chemical energy by introducing rhodopsin into nonphotosynthetic engineered strains could contribute to promoting growth, increasing production and improving cell tolerance of microbial hosts. Gloeorhodopsin (GR) is a PPR from Gloeobacter violaceus PCC 7421. We expressed GR heterologously in Escherichia coli and verified its functional activity. GR could properly function as a light-driven proton pump and its absorption maximum was at 539 nm. We observed that GR was mainly located on the cell membrane and no inclusion body could be found. After increasing expression level by ribosome binding site optimization, intracellular ATP increased, suggesting that GR could supply additional energy to heterologous hosts under given conditions.


Assuntos
Cianobactérias , Rodopsina , Cianobactérias/genética , Cianobactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bombas de Próton , Rodopsina/genética , Rodopsina/metabolismo , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo
12.
J Chem Theory Comput ; 17(4): 2502-2512, 2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33788568

RESUMO

Hopanoids, the bacterial analogues of sterols, are ubiquitous in bacteria and play a significant role in organismal survival under stressful environments. Unlike sterols, hopanoids have a high degree of variation in the size and chemical nature of the substituent attached to the ring moiety, leading to different effects on the structure and dynamics of biological membranes. While it is understood that hopanoids can indirectly tune membrane physical properties, little is known on the role that hopanoids may play in affecting the organization and behavior of bacterial membrane proteins. In this work we used coarse-grained molecular dynamics simulations to characterize the effects of two hopanoids, diploptene (DPT) and bacteriohopanetetrol (BHT), on the oligomerization of proteorhodopsin (PR) in a model membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phophoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (POPG). PR is a bacterial membrane protein that functions as a light-activated proton pump. We chose PR based on its ability to adopt a distribution of oligomeric states in different membrane environments. Furthermore, the efficiency of proton pumping in PR is intimately linked to its organization into oligomers. Our results reveal that both BHT and DPT indirectly affect dimerization by tuning membrane properties in a fashion that is concentration-dependent. Variation in their interaction with PR in the membrane-embedded and the cytoplasmic regions leads to distinctly different effects on the plasticity of the dimer interface. BHT has the ability to intercalate between monomers in the dimeric interface, whereas DPT shifts dimerization interactions via packing of the interleaflet region of the membrane. Our results show a direct relationship between hopanoid structure and lateral organization of PR, providing a first glimpse at how these bacterial analogues to eukaryotic sterols produce very similar biophysical effects within the cell membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Rodopsinas Microbianas/metabolismo , Triterpenos/metabolismo , Proteínas de Bactérias/química , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/química , Dimerização , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Rodopsinas Microbianas/química , Triterpenos/química
13.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33753488

RESUMO

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.


Assuntos
Canais de Cloreto/metabolismo , Cloretos/metabolismo , Rodopsinas Microbianas/metabolismo , Cátions Monovalentes/metabolismo , Canais de Cloreto/isolamento & purificação , Canais de Cloreto/efeitos da radiação , Canais de Cloreto/ultraestrutura , Cristalografia/métodos , Radiação Eletromagnética , Lasers , Simulação de Dinâmica Molecular , Nocardioides , Conformação Proteica em alfa-Hélice/efeitos da radiação , Estrutura Terciária de Proteína/efeitos da radiação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Proteínas Recombinantes/ultraestrutura , Retinaldeído/metabolismo , Retinaldeído/efeitos da radiação , Rodopsinas Microbianas/isolamento & purificação , Rodopsinas Microbianas/efeitos da radiação , Rodopsinas Microbianas/ultraestrutura , Água/metabolismo
14.
Commun Biol ; 4(1): 362, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742139

RESUMO

Microbial rhodopsins are photoreceptive membrane proteins, which are used as molecular tools in optogenetics. Here, a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily. Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases, the ML-based method selected 65 candidate rhodopsins. The wavelengths of 39 of them were able to be experimentally determined by expressing proteins with the Escherichia coli system, and 32 (82%, p = 7.025 × 10-5) actually showed red-shift gains. In addition, four showed red-shift gains >20 nm, and two were found to have desirable ion-transporting properties, indicating that they would be potentially useful in optogenetics. These findings suggest that data-driven ML-based approaches play effective roles in the experimental design of rhodopsin and other photobiological studies. (141/150 words).


Assuntos
Canais Iônicos/metabolismo , Aprendizado de Máquina , Optogenética , Rodopsinas Microbianas/metabolismo , Sequência de Aminoácidos , Teorema de Bayes , Cor , Bases de Dados de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Canais Iônicos/genética , Canais Iônicos/efeitos da radiação , Luz , Estudo de Prova de Conceito , Conformação Proteica em alfa-Hélice , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/efeitos da radiação , Análise de Sequência de Proteína
16.
Nat Plants ; 7(2): 144-151, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33594268

RESUMO

While rhodopsin-based optogenetics has revolutionized neuroscience1,2, poor expression of opsins and the absence of the essential cofactor all-trans-retinal has complicated the application of rhodopsins in plants. Here, we demonstrate retinal production in plants and improved rhodopsin targeting for green light manipulation of plant cells using the Guillardia theta light-gated anion channelrhodopsin GtACR13. Green light induces a massive increase in anion permeability and pronounced membrane potential changes when GtACR1 is expressed, enabling non-invasive manipulation of plant growth and leaf development. Using light-driven anion loss, we could mimic drought conditions and bring about leaf wilting despite sufficient water supply. Expressed in pollen tubes, global GtACR1 activation triggers membrane potential depolarizations due to large anion currents. While global illumination was associated with a reversible growth arrest, local GtACR1 activation at the flanks of the apical dome steers growth direction away from the side with increased anion conductance. These results suggest a crucial role of anion permeability for the guidance of pollen tube tip growth. This plant optogenetic approach could be expanded to create an entire pallet of rhodopsin-based tools4, greatly facilitating dissection of plant ion-signalling pathways.


Assuntos
Optogenética/métodos , Desenvolvimento Vegetal/efeitos dos fármacos , Desenvolvimento Vegetal/fisiologia , Proteobactérias/química , Rodopsinas Microbianas/metabolismo , Tabaco/genética , Tabaco/metabolismo
17.
Phys Chem Chem Phys ; 23(3): 2072-2079, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33433533

RESUMO

We carried out the low-temperature Raman measurement of a sodium pump rhodopsin from Indibacter alkaliphilus (IaNaR) and examined the primary structural change for the light-driven Na+ pump. We observed that photoexcitation of IaNaR produced the distorted 13-cis retinal chromophore in the presence of Na+, while the structural distortion was significantly relaxed in the absence of Na+. This structural difference of the chromophore with/without Na+ was attributed to the Na+ binding to the protein, which alters the active site. Using the spectral sensitivity to the ion binding, we found that IaNaR had a second Na+ binding site in addition to the one already specified on the extracellular surface. To date, the Na+ binding has not been considered as a prerequisite for Na+ transport. However, this study provides insight that the protein structural change induced by the ion binding involved the formation of an R108-D250 salt bridge, which has critical importance in the active transport of Na+.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteroidetes/química , Proteínas de Transporte de Cátions/metabolismo , Rodopsinas Microbianas/metabolismo , Sódio/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Transporte Biológico Ativo , Domínio Catalítico , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/efeitos da radiação , Temperatura Baixa , Cristalografia por Raios X , Diterpenos/química , Conformação Molecular , Mutação , Retinaldeído/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/efeitos da radiação , Análise Espectral Raman
18.
Adv Exp Med Biol ; 1293: 3-19, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33398804

RESUMO

The first light-sensing proteins used in optogenetics were rhodopsins. The word "rhodopsin" originates from the Greek words "rhodo" and "opsis," indicating rose and sight, respectively. Although the classical meaning of rhodopsin is the red-colored pigment in our eyes, the modern meaning of rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial rhodopsins possess 11-cis and all-trans retinal, respectively, to capture light in seven transmembrane α-helices, and photoisomerizations into all-trans and 13-cis forms, respectively, initiate each function. We are able to find ion-transporting proteins in microbial rhodopsins, such as light-gated channels and light-driven pumps, which are the main tools in optogenetics. In this chapter, historical aspects and molecular properties of rhodopsins are introduced. In the first part, "what is rhodopsin?", general introduction of rhodopsin is presented. Then, molecular mechanism of bacteriorodopsin, a light-driven proton pump and the best-studied microbial rhodopsin, is described. In the section of channelrhodopsin, the light-gated ion channel, molecular properties, and several variants are introduced. As the history has proven, understanding the molecular mechanism of microbial rhodopsins is a prerequisite for useful functional design of optogenetics tools in future.


Assuntos
Luz , Rodopsina/metabolismo , Animais , Transporte de Íons/efeitos da radiação , Optogenética/métodos , Rodopsina/genética , Rodopsina/efeitos da radiação , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/efeitos da radiação
19.
Adv Exp Med Biol ; 1293: 55-71, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33398807

RESUMO

Cl--pump rhodopsin is the second discovered microbial rhodopsin. Although its physiological role has not been fully clarified, its functional mechanism has been studied as a model for anion transporters. After the success of neural activation by channel rhodopsin, the first Cl--pump halorhodopsin (HR) had become widely used as a neural silencer. The emergence of artificial and natural anion channel rhodopsins lowered the importance of HRs. However, the longer absorption maxima of approximately 585-600 nm for HRs are still advantageous for applications in mammalian brains and collaborations with neural activators possessing shorter absorption maxima. In this chapter, the variation and functional mechanisms of Cl- pumps are summarized. After the discovery of HR, Cl--pump rhodopsins were confined to only extremely halophilic haloarchaea. However, after 2014, two Cl--pump groups were newly discovered in marine and terrestrial bacteria. These Cl- pumps are phylogenetically distinct from HRs and have unique characteristics. In particular, the most recently identified Cl- pump has close similarity with the H+ pump bacteriorhodopsin and was converted into the H+ pump by a single amino acid replacement.


Assuntos
Cloretos/metabolismo , Bombas de Próton/metabolismo , Prótons , Rodopsinas Microbianas/metabolismo , Animais , Bacteriorodopsinas/metabolismo , Halorrodopsinas/metabolismo , Luz , Bombas de Próton/química , Bombas de Próton/efeitos da radiação , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação
20.
Adv Exp Med Biol ; 1293: 73-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33398808

RESUMO

In these 15 years, researches to control cellular responses by light have flourished dramatically to establish "optogenetics" as a research field. In particular, light-dependent excitation/inhibition of neural cells using channelrhodopsins or other microbial rhodopsins is the most powerful and the most widely used optogenetic technique. New channelrhodopsin-based optogenetic tools having favorable characteristics have been identified from a wide variety of organisms or created through mutagenesis. Despite the great efforts, some neuronal activities are still hard to be manipulated by the channelrhodopsin-based tools, indicating that complementary approaches are needed to make optogenetics more comprehensive. One of the feasible and complementary approaches is optical control of ion channels using photoreceptive proteins other than channelrhodopsins. In particular, animal opsins can modulate various ion channels via light-dependent G protein activation. In this chapter, we summarize how such alternative optogenetic tools work and they will be improved.


Assuntos
Canais Iônicos/metabolismo , Canais Iônicos/efeitos da radiação , Optogenética/métodos , Rodopsinas Microbianas , Animais , Channelrhodopsins/metabolismo , Luz , Neurônios/citologia , Neurônios/metabolismo , Rodopsinas Microbianas/metabolismo
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