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1.
Nat Commun ; 11(1): 5605, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154353

RESUMO

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.


Assuntos
Diester Fosfórico Hidrolases/química , Rodopsinas Microbianas/química , Coanoflagelados/enzimologia , Coanoflagelados/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Domínios Proteicos , Rodopsina , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Relação Estrutura-Atividade
2.
Nature ; 583(7815): 314-318, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499654

RESUMO

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.


Assuntos
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sítios de Ligação , Cristalografia , Elétrons , Transporte de Íons , Isomerismo , Lasers , Prótons , Teoria Quântica , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Sódio/metabolismo , Análise Espectral , Eletricidade Estática , Fatores de Tempo
3.
Proc Natl Acad Sci U S A ; 117(8): 4131-4141, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32034096

RESUMO

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.


Assuntos
Biologia Computacional , Rodopsinas Microbianas/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Fotólise , Conformação Proteica
4.
Sci Rep ; 10(1): 282, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937866

RESUMO

Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seven-transmembrane protein that functions as a light-driven proton (H+) pump with the chromophore retinal. To characterize its vectorial proton transport mechanism, mutational and theoretical investigations were performed for carboxylates in the transmembrane region of RxR and the sequential proton transport steps were revealed as follows: (i) a proton of the retinylidene Schiff base (Lys209) is transferred to the counterion Asp74 upon formation of the blue-shifted M-intermediate in collaboration with Asp205, and simultaneously, a respective proton is released from the proton releasing group (Glu187/Glu197) to the extracellular side, (ii) a proton of Asp85 is transferred to the Schiff base during M-decay, (iii) a proton is taken up from the intracellular side to Asp85 during decay of the red-shifted O-intermediate. This ion transport mechanism of RxR provides valuable information to understand other ion transporters since carboxylates are generally essential for their functions.


Assuntos
Actinobacteria/metabolismo , Rodopsinas Microbianas/metabolismo , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Prótons , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Cloreto de Sódio/química
5.
Sci Rep ; 10(1): 1356, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992768

RESUMO

Proteorhodopsin (PR) is discovered from marine bacteria and it has proton pumping activity from inside to outside of the cell using light energy. In general, PR classified into two groups by the maximum absorption spectra. In this study, we isolated the two of a full sequence of opsin homologues by PCR from the seawater sample near King George Island, Antarctica. One was the same sequence as the first reported GPR (Green-light absorbing PR) from Monterey Bay. Another named HSG119 was a newly discovered sequence which shows high sequence similarity with BPR (Blue-light absorbing PR). HSG119 has an absorption maximum at 493 nm with broader spectrum at pH7.0 and it can pump protons out of the cell membrane. Interestingly, it showed a similar temperature dependence to GPR(Y200N) that isolated near the North pole.


Assuntos
Organismos Aquáticos/fisiologia , Bombas de Próton/genética , Bombas de Próton/metabolismo , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Temperatura , Regiões Antárticas , Mutação , Oceanos e Mares , Processos Fotoquímicos , Bombas de Próton/química , Rodopsinas Microbianas/química , Análise Espectral
6.
Biochemistry ; 59(3): 218-229, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31815443

RESUMO

Microbial rhodopsins, a photoactive membrane protein family, serve as fundamental tools for optogenetics, an innovative technology for controlling biological activities with light. Microbial rhodopsins are widely distributed in nature and have a wide variety of biological functions. Regardless of the many different known types of microbial rhodopsins, only a few of them have been used in optogenetics to control neural activity to understand neural networks. The efforts of our group have been aimed at identifying and characterizing novel rhodopsins from nature and also at engineering novel variant rhodopsins by rational design. On the basis of the molecular and functional characteristics of those novel rhodopsins, we have proposed new rhodopsin-based optogenetics tools to control not only neural activities but also "non-neural" activities. In this Perspective, we introduce the achievements and summarize future challenges in creating optogenetics tools using rhodopsins. The implementation of optogenetics deep inside an in vivo brain is the well-known challenge for existing rhodopsins. As a perspective to address this challenge, we introduce innovative optical illumination techniques using wavefront shaping that can reinforce the low light sensitivity of the rhodopsins and realize deep-brain optogenetics. The applications of our optogenetics tools could be extended to manipulate non-neural biological activities such as gene expression, apoptosis, energy production, and muscle contraction. We also discuss the potentially unlimited biotechnological applications of microbial rhodopsins in the future such as in photovoltaic devices and in drug delivery systems. We believe that advances in the field will greatly expand the potential uses of microbial rhodopsins as optical tools.


Assuntos
Encéfalo/diagnóstico por imagem , Sistemas de Liberação de Medicamentos , Optogenética , Rodopsinas Microbianas/farmacologia , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Rodopsinas Microbianas/química
7.
Biochim Biophys Acta Bioenerg ; 1861(1): 148092, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669491

RESUMO

Although many microbial rhodopsins have been discovered many of organisms in a variety of habitats, little is known about the property and diversity of rhodopsin in flavobacteria. Recent studies discovered that many proteorhodopsin (PR)-like proteins exist in genomes of flavobacteria. Following the isolation of a flavobacterial rhodopsins (FR) from the flavobacteria IMCC1997 from the East Sea of Korea, we characterized its photochemical features. We confirmed that the FR expression is induced by light in the IMCC1997 cell. Upon receiving light energy in vitro, the proton acceptor (D83) and donor (E94) of the FR translocate protons from intracellular to extracellular regions. Compared with proteorhodopsin (PR), the FR from IMCC 1997 cells is very unstable, which may be explained by their primary sequence differences. The ratio of all trans/13-cis retinal conformation does not influence this stability. To measure the stability of FR, we tested heat endurance at 70 °C and found that the heat endurance time of some FR mutants increased. Based upon these results, we found the helix E of this protein to be critical for the unstability of FR.


Assuntos
Proteínas de Bactérias/química , Flavobacterium/química , Temperatura Alta , Rodopsinas Microbianas/química , Estabilidade Proteica , Estrutura Secundária de Proteína
8.
J Phys Chem B ; 123(50): 10631-10641, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31757123

RESUMO

Proteorhodopsin (PR) is a light-driven proton pump that is most notable for ushering in the discovery of an ever-increasing number of microbial retinal proteins that are at the forefront of fields such as optogenetics. Two variants, blue (BPR) and green (GPR) proteorhodopsin, have evolved to harvest light at different depths of the ocean. The color-tuning mechanism in PR is controlled by a single residue at position 105: in BPR it is a glutamine, whereas in GPR it is a leucine. Although the majority of studies on the spectral tuning mechanism in PR have focused on GPR, detailed understanding of the electronic environment responsible for spectral tuning in BPR is lacking. In this work, several BPR models were investigated using quantum mechanics/molecular mechanics (QM/MM) calculations to obtain fundamental insights into the color tuning mechanism of BPR. We find that the molecular mechanism of spectral tuning in BPR depends on two geometric parameters, the bond length alternation and the torsion angle deviation of the all-trans-retinyl chromophore. Both parameters are influenced by the strength of the hydrogen-bonded networks in the chromophore-binding pocket, which shows how BPR is different from other microbial rhodopsins.


Assuntos
Absorção Fisico-Química , Modelos Moleculares , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Domínio Catalítico , Cor , Ligação de Hidrogênio , Teoria Quântica
9.
Phys Chem Chem Phys ; 21(46): 25728-25734, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31720623

RESUMO

Proteorhodopsin (PR) is a proton-pumping rhodopsin, and it is known to exhibit a multi-phasic decay of the excited-state population in the primary process. So far, this complex excited-state decay has been attributed to the branching of the relaxation pathway on the excited-state potential energy surface. However, a recent ultrafast spectroscopic study on a sodium-pumping rhodopsin suggested that such a complex decay may originate from the heterogeneity in the ground state due to the acid-base equilibrium of the counterion of the protonated retinal Schiff base (PRSB). In this study, we studied the excited-state dynamics of PR at pH 11 and 4, in which the counterion of the PRSB, Asp97, is completely deprotonated and protonated, respectively. The obtained time-resolved absorption data revealed that the excited-state lifetime is decisively governed by the protonation state of Asp97, and the photoisomerization of the PRSB chromophore proceeds faster and more efficiently when Asp97 is deprotonated. This conclusion was further supported by high similarity of the excited-state dynamics between PR at pH 4 and the D97N mutant in which Asp97 is replaced with neutral Asn. The results of this study suggest that the protonation state of the PRSB counterion plays a decisive role in determining the excited-state dynamics and the photoisomerization reactivity of rhodopsins in general, by making a significant influence on the exited-state potential energy surface of the PRSB chromophore.


Assuntos
Isomerismo , Rodopsinas Microbianas/química , Concentração de Íons de Hidrogênio , Processos Fotoquímicos , Teoria Quântica
10.
ACS Chem Neurosci ; 10(12): 4768-4775, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31725259

RESUMO

Genetically encoded voltage indicators (GEVIs) allow optical recording of neuronal activities with high spatial resolution. While most existing GEVIs emit in the green range, red-shifted GEVIs are highly sought after because they would enable simultaneous stimulation and recording of neuronal activities when paired with optogenetic actuators, or two-color imaging of signaling and neuronal activities when used along with GFP-based indicators. In this study, we present several improved red-shifted GEVIs based on the electrochromic Förster resonance energy transfer (eFRET) between orange/red fluorescent proteins/dyes and rhodopsin mutants. Through structure-guided mutagenesis and cell-based sensitivity screening, we identified a mutant rhodopsin with a single mutation that exhibited more than 2-fold improvement in voltage sensitivity. Notably, this mutation has been independently discovered by Pieribone et al. ( Pieribone, V. A. et al. Nat Methods 2018 , 15 ( 12 ), 1108 - 1116 ). In cultured rat hippocampal neurons, our sensors faithfully reported action potential waveforms and subthreshold activities. We also demonstrated that this mutation could enhance the sensitivity of hybrid indicators, thus providing insights for future development.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Neurônios/fisiologia , Rodopsinas Microbianas/química , Imagens com Corantes Sensíveis à Voltagem/métodos , Acetabularia/genética , Potenciais de Ação , Substituição de Aminoácidos , Animais , Células Cultivadas , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Proteínas Luminescentes/química , Modelos Moleculares , Mutação de Sentido Incorreto , Optogenética , Mutação Puntual , Conformação Proteica , Engenharia de Proteínas , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodopsinas Microbianas/genética
11.
J Microbiol ; 57(11): 982-990, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31659684

RESUMO

Microalgae and bacteria are known to be closely associated in diverse environments. To isolate dominant bacterial species associated with a green alga, Dunaliella tertiolecta, a photoreactor culture of the microalga was investigated using culture-based and culture-independent approaches. The bacterial community structure of the algal culture showed that the most abundant bacterial species under the culture conditions was related to the genus Winogradskyella. The closely related amplicon sequences, showing ≥ 99.5% 16S rRNA gene sequence similarity to one of the isolates, designated IMCC-33238T, constituted > 49% of the bacterial community and was therefore regarded as the most dominant species in the algal culture. Strain IMCC33238T was characterized by Gramstaining-negative and orange-colored rods. Phylogenetic analyses of the 16S rRNA genes as well as whole genome sequences revealed that strain IMCC33238T belonged to Winogradskyella and shared more than 97.2% 16S rRNA gene sequence similarity with Winogradskyella species. The strain contained iso-C15:1 G, iso-C15:0, iso-C15:0 3-OH, and summed feature 3 (C16:1ω6c and/or C16:1ω7c) as major fatty acids and MK-6 as the predominant quinone. The polar lipids found in strain IMCC33238T were phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. The genome of strain IMCC33238T was 3.37 Mbp in size with 33.9 mol% G + C content and proteorhodopsin. Many genes encoding folate and vitamin production are considered to play an important role in the bacteria-algae interaction. On the basis of phylogenetic and phenotypic characteristics, strain IMCC33238T represents a novel species in the genus Winogradskyella, for which the name Winogradskyella algicola sp. nov. is proposed. The type strain is IMCC33238T (= KACC 21192T = NBRC 113704T).


Assuntos
Clorofíceas/microbiologia , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Filogenia , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , Benzoquinonas/química , DNA Bacteriano/genética , Ácidos Graxos/química , Flavobacteriaceae/fisiologia , Fosfatidiletanolaminas/química , RNA Ribossômico 16S/genética , República da Coreia , Rodopsinas Microbianas/química , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química , Sequenciamento Completo do Genoma
12.
Nature ; 574(7776): 132-136, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554965

RESUMO

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.


Assuntos
Rodopsinas Microbianas/química , Thermoplasmales/química , Bacteriorodopsinas/química , Sítios de Ligação , Cristalografia por Raios X , Microscopia de Força Atômica , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Retinaldeído/química , Rodopsinas Microbianas/ultraestrutura
13.
J Phys Chem Lett ; 10(17): 5117-5121, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31433641

RESUMO

Light energy is first captured in animal and microbial rhodopsins by ultrafast photoisomerization, whose relaxation accompanies protein structural changes for each function. Here, we report a microbial rhodopsin, marine bacterial TAT rhodopsin, that displays no formation of photointermediates at >10-5 s. Low-temperature ultraviolet-visible and Fourier transform infrared spectroscopy revealed that TAT rhodopsin features all-trans to 13-cis photoisomerization like other microbial rhodopsins, but a planar 13-cis chromophore in the primary K intermediate seems to favor thermal back isomerization to the original state without photocycle completion. The molecular mechanism of the early photoreaction in TAT rhodopsin will be discussed.


Assuntos
Rodopsinas Microbianas/química , Espectroscopia de Infravermelho com Transformada de Fourier , Isomerismo , Luz , Proteobactérias/metabolismo , Rodopsinas Microbianas/metabolismo
14.
Science ; 365(6454): 699-704, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31371562

RESUMO

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.


Assuntos
Monitorização Fisiológica/métodos , Neuroimagem/métodos , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Comportamento Animal , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Engenharia Genética , Larva , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Camundongos , Optogenética , Domínios Proteicos , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Natação , Peixe-Zebra
15.
J Phys Chem Lett ; 10(15): 4374-4381, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31313926

RESUMO

Voltage imaging allows mapping of the membrane potential in living cells. Yet, current intensity-based imaging approaches are limited to relative membrane potential changes, missing important information conveyed by the absolute value of the membrane voltage. This challenge arises from various factors affecting the signal intensity, such as concentration, illumination intensity, and photobleaching. Here, we demonstrate electronic preresonance hyperspectral stimulated Raman scattering (EPR-hSRS) for spectroscopic detection of the membrane voltage using a near-infrared-absorbing microbial rhodopsin expressed in E. coli. This newly developed near-infrared active microbial rhodopsin enables electronic preresonance SRS imaging at high sensitivity. By spectral profiling, we identified voltage-sensitive SRS peaks in the fingerprint region in single E. coli cells. These spectral signatures offer a new approach for quantitation of the absolute membrane voltage in living cells.


Assuntos
Rodopsinas Microbianas/química , Análise Espectral Raman/métodos , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Raios Infravermelhos , Potenciais da Membrana , Mutação , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Análise de Célula Única/métodos
16.
J Microbiol ; 57(8): 676-687, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201724

RESUMO

Strain IMCC1322 was isolated from a surface water sample from the East Sea of Korea. Based on 16S rRNA analysis, IMCC1322 was found to belong to the OCS28 sub-clade of SAR116. The cells appeared as short vibrioids in logarithmic-phase culture, and elongated spirals during incubation with mitomycin or in aged culture. Growth characteristics of strain IMCC1322 were further evaluated based on genomic information; proteorhodopsin (PR), carbon monoxide dehydrogenase, and dimethylsulfoniopropionate (DMSP)-utilizing enzymes. IMCC1322 PR was characterized as a functional retinylidene protein that acts as a light-driven proton pump in the cytoplasmic membrane. However, the PR-dependent phototrophic potential of strain IMCC1322 was only observed under CO-inhibited and nutrient-limited culture conditions. A DMSP-enhanced growth response was observed in addition to cultures grown on C1 compounds like methanol, formate, and methane sulfonate. Strain IMCC1322 cultivation analysis revealed biogeochemical processes characteristic of the SAR116 group, a dominant member of the microbial community in euphotic regions of the ocean. The polyphasic taxonomy of strain IMCC1322 is given as Candidatus Puniceispirillum marinum, and was confirmed by chemotaxonomic tests, in addition to 16S rRNA phylogeny and cultivation analyses.


Assuntos
Alphaproteobacteria , RNA Ribossômico 16S/genética , Rodopsinas Microbianas , Água do Mar/microbiologia , Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Alphaproteobacteria/crescimento & desenvolvimento , Alphaproteobacteria/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , DNA Bacteriano/genética , República da Coreia , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Compostos de Sulfônio/metabolismo , Sequenciamento Completo do Genoma/métodos
17.
Biochemistry ; 58(26): 2934-2943, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31150215

RESUMO

Many microorganisms express rhodopsins, pigmented membrane proteins capable of absorbing sunlight and harnessing that energy for important biological functions such as ATP synthesis and phototaxis. Microbial rhodopsins that have been discovered to date are categorized as type-1 rhodopsins. Interestingly, researchers have very recently unveiled a new microbial rhodopsin family named the heliorhodopsins, which are phylogenetically distant from type-1 rhodopsins. Among them, only heliorhodopsin-48C12 (HeR-48C12) from a Gram-positive eubacterium has been photochemically characterized [Pushkarev, A., et al. (2018) Nature 558, 595-599]. In this study, we photochemically characterize a purple-colored heliorhodopsin from Gram-negative eubacterium Bellilinea caldifistulae (BcHeR) as a second example and identify which properties are or are not conserved between BcHeR and HeR-48C12. A series of photochemical measurements revealed several conserved properties between them, including a visible absorption spectrum with a maximum at around 550 nm, the lack of ion-transport activity, and the existence of a second-order O-like intermediate during the photocycle that may activate an unidentified biological function. In contrast, as a property that is not conserved, although HeR-48C12 shows the light adaptation state of retinal, BcHeR showed the same retinal configuration under both dark- and light-adapted conditions. These comparisons of photochemical properties between BcHeR and HeR-48C12 are an important first step toward understanding the nature and functional role of heliorhodopsins.


Assuntos
Proteínas de Bactérias/química , Chloroflexi/química , Rodopsinas Microbianas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Chloroflexi/genética , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/genética , Luz , Processos Fotoquímicos , Filogenia , Rodopsinas Microbianas/genética
18.
J Phys Chem B ; 123(21): 4562-4570, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31050900

RESUMO

The absence of detergent and curvature makes nanodiscs excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid nanodiscs limits their use in the study of the structure, dynamics, and functions of membrane proteins. In this study, we parameterized and optimized the coarse-graining (CG) bead mapping for two differently charged and functionalized copolymers, containing styrene-maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed nanodisc formation (<8 nm diameter) on a time scale of tens of microseconds using molecular dynamics (MD) simulations. Structural models of ∼2.0 or 4.8 kDa PMAQA and ∼2.2 kDa SMAEA polymer-based lipid nanodiscs highlight the importance of the polymer chemical structure, size, and polymer-lipid ratio in the optimization of the nanodisc structure. The ideal spatial arrangement of polymers in nanodiscs, nanodisc size, and thermal stability obtained from our MD simulation correlates well with the experimental observations. The polymer-nanodiscs were tested for the reconstitution of single-pass or multipass transmembrane proteins. We expect this study to be useful in the development of novel polymer-based lipid nanodiscs and for the structural studies of membrane proteins.


Assuntos
Precursor de Proteína beta-Amiloide/química , Integrina beta3/química , Maleatos/química , Nanopartículas/química , Ácidos Polimetacrílicos/química , Poliestirenos/química , Rodopsinas Microbianas/química , Sequência de Aminoácidos , Dimiristoilfosfatidilcolina/química , Humanos , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Nostoc/química , Fosfatidilcolinas/química
19.
Proc Natl Acad Sci U S A ; 116(17): 8342-8349, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30948633

RESUMO

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp-His-Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Rodopsinas Microbianas , Histidina/química , Histidina/metabolismo , Isomerismo , Modelos Moleculares , Subunidades Proteicas/química , Sequências Repetitivas de Aminoácidos , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/ultraestrutura , Triptofano/química , Triptofano/metabolismo
20.
J Phys Chem B ; 123(19): 4242-4250, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30998011

RESUMO

Microbial rhodopsins constitute a key protein family in optobiotechnological applications such as optogenetics and voltage imaging. Spectral tuning of rhodopsins into the deep-red and near-infrared spectral regions is of great demand in such applications because more bathochromic light into the near-infrared range penetrates deeper in living tissue. Recently, retinal analogues have been successfully used in ion transporting and fluorescent rhodopsins to achieve red-shifted absorption, activity, and emission properties. Understanding their photochemical mechanism is essential for further design of appropriate retinal analogues but is yet only poorly understood for most retinal analogue pigments. Here, we report the photoreaction dynamics of red-shifted analogue pigments of the proton pump proteorhodopsin (PR) containing A2 (all- trans-3,4-dehydroretinal), MOA2 (all- trans-3-methoxy-3,4-dehydroretinal), or DMAR (all- trans-3-dimethylamino-16-nor-1,2,3,4-didehydroretinal), utilizing femto- to submillisecond transient absorption spectroscopy. We found that the A2 analogue photoisomerizes in 1.4, 3.0, and/or 13 ps upon 510 nm light illumination, which is comparable to the native retinal (A1) in PR. On the other hand, the deprotonation of the A2 pigment Schiff base was observed with a dominant time constant of 67 µs, which is significantly slower than the A1 pigment. In the MOA2 pigment, no isomerization or photoproduct formation was detected upon 520 nm excitation, implying that all the excited molecules returned to the initial ground state in 2.0 and 4.2 ps. The DMAR pigment showed very slow excited state dynamics similar to the previously studied MMAR pigment, but only very little photoproduct was formed. The low efficiency of the photoproduct formation likely is the reason why DMAR analogue pigments of PR showed very weak proton pumping activity.


Assuntos
Retinaldeído/análogos & derivados , Rodopsinas Microbianas/química , Luz , Retinaldeído/efeitos da radiação , Rodopsinas Microbianas/efeitos da radiação
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