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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
J Biomol NMR ; 73(1-2): 49-58, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30719609

RESUMO

The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR. We present the novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Previously, the biosynthesis of a retinal precursor, ß-carotene, has been introduced into many different organisms. We extended this system to the prototrophic E. coli expression strain BL21 in conjunction with the inducible expression of a ß-dioxygenase and proteo-opsin. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern.


Assuntos
Isótopos de Carbono , Retinaldeído/biossíntese , Rodopsinas Microbianas/química , Escherichia coli/química , Glucose/metabolismo , Marcação por Isótopo , Opsinas , Retinaldeído/metabolismo , Rodopsinas Microbianas/economia , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/fisiologia , beta Caroteno/metabolismo
9.
J Chem Phys ; 150(5): 055101, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736678

RESUMO

Although the two membrane proteins, thermophilic rhodopsin (TR) and xanthorhodopsin (XR), share a high similarity in amino-acid sequence and an almost indistinguishable three-dimensional structure, TR is much more thermostable than XR. This is counterintuitive also because TR possesses only a smaller number of intramolecular hydrogen bonds (HBs) than XR. Here we investigate physical origins of the remarkable difference between XR and TR in the stability. Our free-energy function (FEF) is improved so that not only the portion within the transmembrane (TM) region but also the extracellular and intracellular portions within the water-immersed (WI) regions can be considered in assessing the stability. The assessment is performed on the basis of the FEF change upon protein folding, which is calculated for the crystal structure of XR or TR. Since the energetics within the TM region is substantially different from that within the WI regions, we determine the TM and WI portions of XR or TR by analyzing the distribution of water molecules using all-atom molecular dynamics simulations. The energetic component of the FEF change consists of a decrease in energy arising from the formation of intramolecular HBs and an increase in energy caused by the break of protein-water HBs referred to as "energetic dehydration penalty." The entropic component is a gain of the translational, configurational entropies of hydrocarbon groups within the lipid bilayer and of water molecules. The entropic component is calculated using the integral equation theory combined with our morphometric approach. The energetic one is estimated by a simple but physically reasonable method. We show that TR is much more stable than XR for the following reasons: The decrease in energy within the TM region is larger, and the energetic dehydration penalty within the WI regions is smaller, leading to higher energetic stabilization, and tighter packing of side chains accompanying the association of seven helices confers higher entropic stabilization on TR.


Assuntos
Proteínas de Bactérias/química , Rodopsinas Microbianas/química , Termodinâmica , Sequência de Aminoácidos , Bacteroidetes/química , Entropia , Ligações de Hidrogênio , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Estabilidade Proteica , Estatística como Assunto , Thermus thermophilus/química , Água/química
10.
Methods Enzymol ; 615: 131-175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30638529

RESUMO

We outline the physical properties of hydration water that are captured by Overhauser Dynamic Nuclear Polarization (ODNP) relaxometry and explore the insights that ODNP yields about the water and the surface that this water is coupled to. As ODNP relies on the pairwise cross-relaxation between the electron spin of a spin probe and a proton nuclear spin of water, it captures the dynamics of single-particle diffusion of an ensemble of water molecules moving near the spin probe. ODNP principally utilizes the same physics as other nuclear magnetic resonance (NMR) relaxometry (i.e., relaxation measurement) techniques. However, in ODNP, electron paramagnetic resonance (EPR) excites the electron spins probes and their high net polarization acts as a signal amplifier. Furthermore, it renders ODNP parameters highly sensitive to water moving at rates commensurate with the EPR frequency of the spin probe (typically 10GHz). Also, ODNP selectively enhances the NMR signal contributions of water moving within close proximity to the spin label. As a result, ODNP can capture ps-ns movements of hydration waters with high sensitivity and locality, even in samples with protein concentrations as dilute as 10 µM. To date, the utility of the ODNP technique has been demonstrated for two major applications: the characterization of the spatial variation in the properties of the hydration layer of proteins or other surfaces displaying topological diversity, and the identification of structural properties emerging from highly disordered proteins and protein domains. The former has been shown to correlate well with the properties of hydration water predicted by MD simulations and has been shown capable of evaluating the hydrophilicity or hydrophobicity of a surface. The latter has been demonstrated for studies of an interhelical loop of proteorhodopsin, the partial structure of α-synuclein embedded at the lipid membrane surface, incipient structures adopted by tau proteins en route to fibrils, and the structure and hydration profile of a transmembrane peptide. This chapter focuses on offering a mechanistic understanding of the ODNP measurement and the molecular dynamics encoded in the ODNP parameters. In particular, it clarifies how the electron-nuclear dipolar coupling encodes information about the molecular dynamics in the nuclear spin self-relaxation and, more importantly, the electron-nuclear spin cross-relaxation rates. The clarification of the molecular dynamics underlying ODNP should assist in establishing a connection to theory and computer simulation that will offer far richer interpretations of ODNP results in future studies.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Proteínas/química , Água/química , Animais , Bactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos de Membrana/química , Rodopsinas Microbianas/química , Marcadores de Spin , alfa-Sinucleína/química
11.
J Biol Chem ; 294(3): 794-804, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30455349

RESUMO

Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.


Assuntos
Actinomycetales/química , Canais de Cloreto/química , Rodopsinas Microbianas/química , Cristalografia por Raios X , Domínios Proteicos
12.
PLoS One ; 13(12): e0209506, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30586409

RESUMO

Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified ß-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore.


Assuntos
Bacteriorodopsinas/química , Rodopsinas Microbianas/química , Luz , Mutação , Optogenética/métodos , Retinaldeído/análogos & derivados , Retinaldeído/química , Rodopsinas Microbianas/genética , Espalhamento de Radiação , Análise Espectral Raman , Estereoisomerismo
13.
J Phys Chem Lett ; 9(22): 6431-6436, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30351947

RESUMO

Heliorhodopsins (HeRs) are a new category of retinal-bound proteins recently discovered through functional metagenomics analysis that exhibit obvious differences from type-1 microbial rhodopsins. We conducted the first detailed structural characterization of the retinal chromophore in HeRs using resonance Raman spectroscopy. The observed spectra clearly show that the Schiff base of the chromophore is protonated and forms a strong hydrogen bond to a species other than a water molecule, highly likely a counterion residue. The vibrational mode of the Schiff base of HeRs exhibits similarities with that of photosensory microbial rhodopsins, that is consistent with the previous proposal that HeRs function as photosensors. We also revealed unusual spectral features of the in-plane chain vibrations of the chromophore, suggesting an unprecedented geometry of the Schiff base caused by a difference in the retinal pocket structure of HeRs. These data demonstrate structural characteristics of the photoreceptive site in this novel type of rhodopsin family.


Assuntos
Proteínas Arqueais/química , Rodopsinas Microbianas/química , Bases de Schiff/química , Halobacterium salinarum/química , Ligações de Hidrogênio , Estrutura Molecular , Conformação Proteica , Análise Espectral Raman/métodos , Thermoplasmales/química , Vibração
14.
J Phys Chem Lett ; 9(22): 6469-6474, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30376338

RESUMO

Near-infrared (NIR)-driven rhodopsins are of great interest in optogenetics and other optobiotechnological developments such as artificial photosynthesis and deep-tissue voltage imaging. Here we report that the proton pump proteorhodopsin (PR) containing a NIR-active retinal analogue (PR:MMAR) exhibits intense NIR fluorescence at a quantum yield of 3.3%. This is 130 times higher than native PR ( Lenz , M. O. ; Biophys J. 2006 , 91 , 255 - 262 ) and 3-8 times higher than the QuasAr and PROPS voltage sensors ( Kralj , J. ; Science 2011 , 333 , 345 - 348 ; Hochbaum , D. R. ; Nat. Methods 2014 , 11 , 825 - 833 ). The NIR fluorescence strongly depends on the pH in the range of 6-8.5, suggesting potential application of MMAR-binding proteins as ultrasensitive NIR-driven pH and/or voltage sensors. Femtosecond transient absorption spectroscopy showed that upon near-IR excitation, PR:MMAR features an unusually long fluorescence lifetime of 310 ps and the absence of isomerized photoproducts, consistent with the high fluorescence quantum yield. Stimulated Raman analysis indicates that the NIR-absorbing species develops upon protonation of a conserved aspartate, which promotes charge delocalization and bond length leveling due to an additional methylamino group in MMAR, in essence providing a secondary protonated Schiff base. This results in much smaller bond length alteration along the conjugated backbone, thereby conferring significant single-bond character to the C13═C14 bond and structural deformation of the chromophore, which interferes with photoinduced isomerization and extends the lifetime for fluorescence. Hence, our studies allow for a molecular understanding of the relation between absorption/emission wavelength, isomerization, and fluorescence in PR:MMAR. As acidification enhances the resonance state, this explains the strong pH dependence of the NIR emission.


Assuntos
Retinaldeído/análogos & derivados , Rodopsinas Microbianas/química , Fluorescência , Concentração de Íons de Hidrogênio , Prótons , Bases de Schiff/química , Análise Espectral Raman
15.
Sci Rep ; 8(1): 15580, 2018 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-30349075

RESUMO

The light-dependent ion-transport function of microbial rhodopsin has been widely used in optogenetics for optical control of neural activity. In order to increase the variety of rhodopsin proteins having a wide range of absorption wavelengths, the light absorption properties of various wild-type rhodopsins and their artificially mutated variants were investigated in the literature. Here, we demonstrate that a machine-learning-based (ML-based) data-driven approach is useful for understanding and predicting the light-absorption properties of microbial rhodopsin proteins. We constructed a database of 796 proteins consisting of microbial rhodopsin wildtypes and their variants. We then proposed an ML method that produces a statistical model describing the relationship between amino-acid sequences and absorption wavelengths and demonstrated that the fitted statistical model is useful for understanding colour tuning rules and predicting absorption wavelengths. By applying the ML method to the database, two residues that were not considered in previous studies are newly identified to be important to colour shift.


Assuntos
Fenômenos Químicos , Cor , Proteínas Mutantes/química , Rodopsinas Microbianas/química , Aprendizado de Máquina , Modelos Estatísticos , Proteínas Mutantes/genética , Rodopsinas Microbianas/genética
16.
Biophys J ; 115(7): 1240-1250, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30219284

RESUMO

Proteorhodopsin (PR) is a microbial proton pump that is ubiquitous in marine environments and may play an important role in the oceanic carbon cycle. Photoisomerization of the retinal chromophore in PR leads to a series of proton transfers between specific acidic amino acid residues and the Schiff base of retinal, culminating in a proton motive force to facilitate ATP synthesis. The proton donor in a similar retinal protein, bacteriorhodopsin, acts as a latch to allow the influx of bulk water. However, it is unclear if the proton donor in PR, E108, utilizes the same latch mechanism to become internally hydrated. Here, we used molecular dynamics simulations to model the changes in internal hydration of the blue variant of PR during photoactivation with the proton donor in protonated and deprotonated states. We find that there is a stark contrast in the levels of internal hydration of the cytoplasmic half of PR based on the protonation state of E108. Instead of a latch mechanism, deprotonation of E108 acts as a gate, taking advantage of a nearby polar residue (S61) to promote the formation of a stable water wire from bulk cytoplasm to the retinal-binding pocket over hundreds of nanoseconds. No large-scale conformational changes occur in PR over the microsecond timescale. This subtle yet clear difference in the effect of deprotonation of the proton donor in PR may help explain why the photointermediates that involve the proton donor (i.e., M and N states) have timescales that are orders of magnitude different from the archaeal proton pump, bacteriorhodopsin. In general, our study highlights the importance of understanding how structural fluctuations lead to differences in the way that retinal proteins accomplish the same task.


Assuntos
Bombas de Próton/química , Bombas de Próton/metabolismo , Prótons , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Regulação Alostérica , Sítios de Ligação , Citoplasma/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Retinaldeído/metabolismo
17.
FEBS Lett ; 592(18): 3054-3061, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30098005

RESUMO

Proteorhodopsin (PR) is a microbial rhodopsin functioning as a light-driven proton pump in aquatic bacteria. We performed low-temperature Raman measurements of PR to obtain the structure of the primary photoproduct, the K intermediate (PRK ). PRK showed the hydrogen-out-of-plane modes that are much less intense than those of bacteriorhodopsin as the prototypical light-driven proton pump from haloarchaea. The present results reveal the significantly relaxed chromophore structure in PRK , which can be coupled to the slow kinetics of the K intermediate. This structure suggests that PR transports protons using the small energy storage within the chromophore at the start of its photocycle.


Assuntos
Temperatura Baixa , Luz , Rodopsinas Microbianas/química , Análise Espectral Raman/métodos , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Cinética , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Conformação Proteica/efeitos da radiação , Bombas de Próton/química , Bombas de Próton/metabolismo , Rodopsinas Microbianas/metabolismo
18.
ACS Synth Biol ; 7(9): 2282-2295, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30053372

RESUMO

In nature, enzymatic reaction cascades, i.e., realized in metabolic networks, operate with unprecedented efficacy, with the reactions often being spatially and temporally orchestrated. The principle of "learning from nature" has in recent years inspired the setup of synthetic reaction cascades combining biocatalytic reaction steps to artificial cascades. Hereby, the spatial organization of multiple enzymes, e.g., by coimmobilization, remains a challenging task, as currently no generic principles are available that work for every enzyme. We here present a tunable, genetically programmed coimmobilization strategy that relies on the fusion of a coiled-coil domain as aggregation inducing-tag, resulting in the formation of catalytically active inclusion body coimmobilizates (Co-CatIBs). Coexpression and coimmobilization was proven using two fluorescent proteins, and the strategy was subsequently extended to two enzymes, which enabled the realization of an integrated enzymatic two-step cascade for the production of (1 R,2 R)-1-phenylpropane-1,2-diol (PPD), a precursor of the calicum channel blocker diltiazem. In particular, the easy production and preparation of Co-CatIBs, readily yielding a biologically produced enzyme immobilizate renders the here presented strategy an interesting alternative to existing cascade immobilization techniques.


Assuntos
Enzimas Imobilizadas/metabolismo , Corpos de Inclusão/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Liases/química , Aldeído Liases/genética , Aldeído Liases/metabolismo , Biocatálise , Cromatografia Líquida de Alta Pressão , Enzimas Imobilizadas/química , Escherichia coli/metabolismo , Propanóis/análise , Propanóis/química , Propanóis/metabolismo , Pseudomonas fluorescens/enzimologia , Ralstonia/enzimologia , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo
19.
Biochemistry ; 57(33): 5041-5049, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30036039

RESUMO

Rhodopsins are heptahelical transmembrane photoactive protein families: type 1 (microbial rhodopsins) and type 2 (animal rhodopsins). Both families share similar topologies and chromophore retinal, which is linked covalently as a protonated Schiff base to a Lys at the transmembrane 7 helix. Recently, through functional metagenomics analysis, we reported an unnoticed diverse family, heliorhodopsins (HeRs), which are abundant and distributed globally in archaea, bacteria, eukarya, and viruses. The sequence identity is <15% between HeRs and type 1 rhodopsins, so that many aspects of the molecular properties of HeRs remain unknown. Herein, to gain information about the residues responsible for the interaction with the chromophore, we applied Ala scanning to 30 candidate residues in HeR 48C12. As a result, 12 mutants showed no absorption change, eight exhibited a spectral blue-shift, six exhibited a spectral red-shift, and four did not form a pigment. R104, Y108, G145, and K241 play crucial roles in pigment formation. A combination of single mutants successfully engineered pigments absorbing at 523 nm (S112A/M141A) and 571 nm (H80A/S237A), covering more than ∼50 nm. These results provide fundamental knowledge about the molecular properties of HeRs.


Assuntos
Rodopsinas Microbianas/genética , Sítios de Ligação , Cor , Escherichia coli/genética , Luz , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Retinaldeído/química , Retinaldeído/metabolismo , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , Bases de Schiff/química
20.
J Phys Chem B ; 122(27): 6945-6953, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29893559

RESUMO

Thermophilic rhodopsin (TR) is a light-driven proton pump from the extreme thermophile Thermus thermophilus JL-18. Previous studies on TR solubilized with detergent showed that the protein exhibits high thermal stability and forms a trimer at room temperature but irreversibly dissociates into monomers when incubated at physiological temperature (75 °C). In the present study, we used resonance Raman (RR) spectroscopy, solid-state NMR spectroscopy, and high-speed atomic force microscopy to analyze the oligomeric structure of TR in a lipid environment. The obtained spectra and microscopic images demonstrate that TR adopts a pentameric form in a lipid environment and that this assembly is stable at the physiological temperature, in contrast to the behavior of the protein in the solubilized state. These results indicate that the thermal stability of the oligomeric assembly of TR is higher in a lipid environment than in detergent micelles. The observed RR spectra also showed that the retinal chromophore is strongly hydrogen bonded to an internal water molecule via a protonated Schiff base, which is characteristic of proton-pumping rhodopsins. The obtained data strongly suggest that TR functions in the pentameric form at physiological temperature in the extreme thermophile T. thermophilus JL-18. We utilized the high thermal stability of the monomeric form of solubilized TR and here report the first RR spectra of the monomeric form of a microbial rhodopsin. The observed RR spectra revealed that the monomerization of TR alters the chromophore structure: there are changes in the bond alternation of the polyene chain and in the hydrogen-bond strength of the protonated Schiff base. The present study revealed the high thermal stability of oligomeric assemblies of TR in the lipid environment and suggested the importance of using TR embedded in lipid membrane for elucidation of its functional mechanism.


Assuntos
Lipídeos/química , Rodopsinas Microbianas/química , Thermus thermophilus/metabolismo , Ligações de Hidrogênio , Microscopia de Força Atômica , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Análise Espectral Raman , Temperatura Ambiente , Água/metabolismo
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