RESUMEN
Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.
RESUMEN
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.
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Rodopsinas Microbianas/química , Thermoplasmales/química , Bacteriorodopsinas/química , Sitios de Unión , Cristalografía por Rayos X , Microscopía de Fuerza Atómica , Modelos Moleculares , Pliegue de Proteína , Multimerización de Proteína , Retinaldehído/química , Rodopsinas Microbianas/ultraestructuraRESUMEN
Microbial rhodopsins are light-receptive proteins with various functions triggered by the photoisomerization of the retinal chromophore from the all-trans to 13-cis configuration. A retinal chromophore is covalently bound to a lysine residue in the middle of the seventh transmembrane helix via a protonated Schiff base. Bacteriorhodopsin (BR) variants lacking a covalent bond between the side chain of Lys-216 and the main chain formed purple pigments and exhibited a proton-pumping function. Therefore, the covalent bond linking the lysine residue and the protein backbone is not considered a prerequisite for microbial rhodopsin function. To further examine this hypothesis regarding the role of the covalent bond at the lysine side chain for rhodopsin functions, we investigated K255G and K255A variants of sodium-pumping rhodopsin, Krokinobacter rhodopsin 2 (KR2), with an alkylamine retinal Schiff base (prepared by mixing ethyl- or n-propylamine and retinal (EtSB or nPrSB)). The KR2 K255G variant incorporated nPrSB and EtSB as similarly to the BR variants, whereas the K255A variant did not incorporate these alkylamine Schiff bases. The absorption maximum of K255G + nPrSB was 524-516 nm, which was close to the 526 nm absorption maximum of the wild-type + all-trans retinal (ATR). However, the K255G + nPrSB did not exhibit any ion transport activity. Since the KR2 K255G variant easily released nPrSB during light illumination and did not form an O intermediate, we concluded that a covalent bond at Lys-255 is important for the stable binding of the retinal chromophore and formation of an O intermediate to achieve light-driven Na+ pump function in KR2.
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Flavobacteriaceae , Rodopsina , Rodopsina/química , Bases de Schiff/química , Lisina/metabolismo , Flavobacteriaceae/metabolismo , Transporte Iónico , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Sodio/metabolismo , LuzRESUMEN
Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins. It was reported that a sodium-pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2), showed a color change from red to purple upon dehydration under crystalline conditions. Here, we applied comprehensive visible and IR absorption spectroscopy and resonance Raman spectroscopy to KR2 in liposomes under hydration-controlled conditions. A large increase in the hydrogen-out-of-plane (HOOP) vibration at 947 (H-C11=C12-H Au mode) and moderate increases at 893 (C7-H and C10-H) and 808 (C14-H) cm-1 were observed under dehydrated conditions, which were assigned by using systematically deuterated retinal. Moreover, the Asn variant at Asp116, which functions as a counter ion for the protonated retinal Schiff base (PRSB), caused a large redshift in the absorption maximum and constitutive increase in the HOOP modes under hydrated and dehydrated conditions. The protonation of a counter ion at Asp116 clearly causes a redshift in the absorption maximum as the all-trans retinal chromophore twists upon dehydration. Namely, the results strongly suggested that water molecules are important for maintaining the hydrogen-bonding network at the PRSB and deprotonation state of Asp116 in KR2.
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Retinaldehído , Rodopsina , Humanos , Retinaldehído/química , Deshidratación , Hidrógeno , AguaRESUMEN
Heliorhodopsins (HeRs), a recently discovered family of rhodopsins, have an inverted membrane topology compared to animal and microbial rhodopsins. The slow photocycle of HeRs suggests a light-sensor function, although the actual function remains unknown. Although HeRs exhibit no specific binding of monovalent cations or anions, recent ATR-FTIR spectroscopy studies have demonstrated the binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR) and 48C12. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding, suggesting a possible modification of the HeR function by Zn2+. The present study shows that Zn2+-binding lowers the thermal stability of TaHeR, and slows back proton transfer to the retinal Schiff base (M decay) during its photocycle. Zn2+-binding was similarly observed for a TaHeR opsin that lacks the retinal chromophore. We then studied the Zn2+-binding site by means of the ATR-FTIR spectroscopy of site-directed mutants. Among five and four mutants of His and Asp/Glu, respectively, only E150Q exhibited a completely different spectral feature of the α-helix (amide-I) in ATR-FTIR spectroscopy, suggesting that E150 is responsible for Zn2+-binding. Molecular dynamics (MD) simulations built a coordination structure of Zn2+-bound TaHeR, where E150 and protein bound water molecules participate in direct coordination. It was concluded that the specific binding site of Zn2+ is located at the cytoplasmic side of TaHeR, and that Zn2+-binding affects the structure and structural dynamics, possibly modifying the unknown function of TaHeR.
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Protones , Rodopsinas Microbianas , Rodopsinas Microbianas/química , Sitios de Unión , ZincRESUMEN
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.
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Canales Iónicos/metabolismo , Canales Iónicos/efectos de la radiación , Optogenética/métodos , Rodopsinas Microbianas , Animales , Channelrhodopsins/metabolismo , Luz , Neuronas/citología , Neuronas/metabolismo , Rodopsinas Microbianas/metabolismoRESUMEN
Canonical K+ channels are tetrameric and highly K+-selective, whereas two-pore-domain K+ (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na+ and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K+-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K+ ions, WT and both variants exhibit an amide-I band at 1680 cm-1 This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K+, a feature very similar to that of the canonical K+ channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K+ affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm-1 band upon changes from K+ to Rb+ or Cs+ conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K+ and Na+ solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K+ channels.
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Canales de Potasio de Dominio Poro en Tándem/metabolismo , Potasio/metabolismo , Animales , Fenómenos Biofísicos , Cationes , Enlace de Hidrógeno , Liposomas , Ratones , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Canales de Potasio de Dominio Poro en Tándem/química , Conformación Proteica , Teoría Cuántica , Sodio/metabolismo , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Ciliary opsins were classically thought to function only in vertebrates for vision, but they have also been identified recently in invertebrates for non-visual photoreception. Larvae of the annelid Platynereis dumerilii are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. Platynereis c-opsin is suggested to relay light signals for melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades and that it can directly bind exogenous all-trans-retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require a supply of 11-cis-retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all-trans-retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11-cis-retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the Platynereis brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of Platynereis to enable detection of ambient UV light. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model.
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Proteínas del Tejido Nervioso/metabolismo , Opsinas/metabolismo , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Poliquetos/fisiología , Sistemas de Mensajero Secundario/efectos de la radiación , Zooplancton/fisiología , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Cilios/metabolismo , Cilios/efectos de la radiación , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Lisina/química , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Oocitos/efectos de la radiación , Opsinas/química , Opsinas/genética , Técnicas de Placa-Clamp , Células Fotorreceptoras de Invertebrados/metabolismo , Filogenia , Poliquetos/efectos de la radiación , Estabilidad Proteica/efectos de la radiación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Retinaldehído/química , Retinaldehído/metabolismo , Estereoisomerismo , Rayos Ultravioleta , Xenopus , Zooplancton/efectos de la radiaciónRESUMEN
Long-wavelength-sensitive (LWS) pigment possesses a chloride binding site in its protein moiety. The binding of chloride alters the absorption spectra of LWS; this is known as the chloride effect. Although the two amino acid substitutions of His197 and Lys200 influence the chloride effect, the molecular mechanism of chloride binding, which underlies the spectral tuning, has yet to be clarified. In this study, we applied ATR-FTIR spectroscopy to monkey green (MG) pigment to gain structural information of the chloride binding site. The results suggest that chloride binding stabilizes the ß-sheet structure on the extracellular side loop with perturbation of the retinal polyene chain, promotes a hydrogen bonding exchange with the hydroxyl group of Tyr, and alters the protonation state of carboxylate. Combining with the results of the binding analyses of various anions (Br-, I- and NO3-), our findings suggest that the anion binding pocket is organized for only Cl- (or Br-) to stabilize conformation around the retinal chromophore, which is functionally relevant with absorbing long wavelength light.
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Cloruros/química , Pigmentos Retinianos/química , Animales , Aniones/química , Sitios de Unión , Cloruros/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Enlace de Hidrógeno , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Pigmentos Retinianos/genética , Pigmentos Retinianos/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.
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Mamíferos/genética , Mamíferos/metabolismo , Retinaldehído/química , Opsinas de Bastones/química , Opsinas de Bastones/genética , Secuencia de Aminoácidos , Animales , Evolución Molecular , Femenino , Galago , Variación Genética , Humanos , Anfioxos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/metabolismo , Oocitos/efectos de la radiación , Papio anubis , Células Fotorreceptoras de Vertebrados/química , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Filogenia , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efectos de la radiación , Células Ganglionares de la Retina/química , Células Ganglionares de la Retina/efectos de la radiación , Pigmentos Retinianos/química , Pigmentos Retinianos/genética , Pigmentos Retinianos/efectos de la radiación , Opsinas de Bastones/efectos de la radiación , Saimiri , Bases de Schiff/química , Homología de Secuencia de Aminoácido , Arañas , XenopusRESUMEN
Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
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Chlamydomonas reinhardtii , Luz , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Secuencia de Aminoácidos , Activación del Canal Iónico , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/genética , Rodopsina/genéticaRESUMEN
V-ATPase is an ATP-driven rotary motor that vectorially transports ions. Together with F-ATPase, a homologous protein, several models on the ion transport have been proposed, but their molecular mechanisms are yet unknown. V-ATPase from Enterococcus hirae forms a large supramolecular protein complex (total molecular weight: ~700,000) and physiologically transports Na⺠and Li⺠across a hydrophobic lipid bilayer. Stabilization of these cations in the binding site has been discussed on the basis of X-ray crystal structures of a membrane-embedded domain, the K-ring (Na⺠and Li⺠bound forms). Sodium or lithium ion binding-induced difference FTIR spectra of the intact E. hirae V-ATPase have been measured in aqueous solution at physiological temperature. The results suggest that sodium or lithium ion binding induces the deprotonation of Glu139, a hydrogen-bonding change in the tyrosine residue and rigid α-helical structures. Identical difference FTIR spectra between the entire V-ATPase complex and K-ring strongly suggest that protein interaction with the I subunit does not cause large structural changes in the K-ring. This result supports the previously proposed Na⺠transport mechanism by V-ATPase stating that a flip-flop movement of a carboxylate group of Glu139 without large conformational changes in the K-ring accelerates the replacement of a Na⺠ion in the binding site. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.
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ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Sitios de Unión , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Enterococcus/química , Enterococcus/metabolismo , Enlace de Hidrógeno , Litio/química , Litio/metabolismo , Modelos Moleculares , Sodio/química , Sodio/metabolismo , Espectrofotometría Infrarroja/métodosRESUMEN
Microbial rhodopsins are classified into type-I rhodopsins, which utilize light energy to perform wide varieties of function, such as proton pumping, ion pumping, light sensing, cation channels, and so on. The crystal structures of several type-I rhodopsins were solved and the molecular mechanisms have been investigated based on the atomic structures. However, the crystal structures of proteins of interest are not always available and the basic architectures are sometimes quite similar, which obscures how the proteins achieve different functions. Stimulus-induced difference FTIR spectroscopy is a powerful tool to detect minute structural changes providing a clue for elucidating the molecular mechanisms. In this review, the studies on type-I rhodopsins from fungi and marine bacteria, whose crystal structures have not been solved yet, were summarized. Neurospora rhodopsin and Leptosphaeria rhodopsin found from Fungi have sequence similarity. The former has no proton pumping function, while the latter has. Proteorhodopsin is another example, whose proton pumping machinery is altered at alkaline and acidic conditions. We described how the structural changes of protein were different and how water molecules were involved in them. We reviewed the results on dynamics of the internal water molecules in pharaonis halorhodopsin as well. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
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Bacteriorodopsinas/química , Halorrodopsinas/química , Rodopsina/química , Rodopsinas Sensoriales/química , Agua/química , Bacteriorodopsinas/metabolismo , Euryarchaeota/química , Euryarchaeota/fisiología , Halorrodopsinas/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Transporte Iónico , Luz , Fototransducción , Modelos Moleculares , Neurospora/química , Neurospora/fisiología , Conformación Proteica , Rodopsina/metabolismo , Rodopsinas Microbianas , Saccharomycetales/química , Saccharomycetales/fisiología , Rodopsinas Sensoriales/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with (15)N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI-HtrI complex.
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Halorrodopsinas/química , Histidina/química , Rodopsinas Sensoriales/química , Halobacterium salinarum/metabolismo , Halorrodopsinas/genética , Halorrodopsinas/metabolismo , Mutagénesis Sitio-Dirigida , Isótopos de Nitrógeno , Protones , Bases de Schiff/química , Rodopsinas Sensoriales/genética , Rodopsinas Sensoriales/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Chirped-pulse upconversion technique has been applied to attenuated total reflectance (ATR) infrared spectroscopy. An extremely broadband infrared pulse was sent to an ATR diamond prism and the reflected pulse was converted to the visible by using four-wave mixing in krypton gas. Absorption spectra of liquids in the range from 200 to 5500 cm(-1) were measured with a visible spectrometer on a single-shot basis. The system was applied to observe the dynamics of exchanging process of two solvents, water and acetone, which give clear vibrational spectral contrast. We observed that the exchange was finished within â¼ 10 ms.
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Luz , Acetona/química , Soluciones , Espectroscopía Infrarroja por Transformada de Fourier , Agua/químicaRESUMEN
Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length of side chains determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.
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Protones , Rodopsinas Microbianas , Thermoplasmales , Concentración de Iones de Hidrógeno , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Bases de Schiff/química , Espectroscopía Infrarroja por Transformada de Fourier , Agua/química , Thermoplasmales/genética , Thermoplasmales/metabolismo , Mutación , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
Heliorhodopsin (HeR) is a new rhodopsin family discovered in 2018 through functional metagenomic analysis. Similar to microbial rhodopsins, HeR has an all-trans retinal chromophore, and its photoisomerization to the 13-cis form triggers a relatively slow photocycle with sequential intermediate states (K, M, and O intermediates). The O intermediate has a relatively long lifetime and is a putative active state for transferring signals or regulating enzymatic reactions. Although the first discovered HeR, 48C12, was found in bacteria and the second HeR (TaHeR) was found in archaea, their key amino acid residues and molecular architectures have been recognized to be well conserved. Nevertheless, the rise and decay kinetics of the O intermediate are faster in 48C12 than in TaHeR. Here, using a new infrared spectroscopic technique with quantum cascade lasers, we clarified that the hydrogen bond between transmembrane helices (TM) 3 and 4 is essential for the altered O kinetics (Ser112 and Asn138 in 48C12). Interconverting mutants of 48C12 and TaHeR clearly revealed that the hydrogen bond is important for regulating the dynamics of the O intermediate. Overall, our study sheds light on the importance of the hydrogen bond between TM3 and TM4 in heliorhodopsins, similar to the DC gate in channelrhodopsins.
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Enlace de Hidrógeno , Cinética , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/genética , Serina/química , Serina/metabolismo , Asparagina/química , Asparagina/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
TRP channels are important pharmacological targets in physiopathology. TRPV2 plays distinct roles in cardiac and neuromuscular function, immunity, and metabolism, and is associated with pathologies like muscular dystrophy and cancer. However, TRPV2 pharmacology is unspecific and scarce at best. Using in silico similarity-based chemoinformatics we obtained a set of 270 potential hits for TRPV2 categorized into families based on chemical nature and similarity. Docking the compounds on available rat TRPV2 structures allowed the clustering of drug families in specific ligand binding sites. Starting from a probenecid docking pose in the piperlongumine binding site and using a Gaussian accelerated molecular dynamics approach we have assigned a putative probenecid binding site. In parallel, we measured the EC50 of 7 probenecid derivatives on TRPV2 expressed in Pichia pastoris using a novel medium-throughput Ca2+ influx assay in yeast membranes together with an unbiased and unsupervised data analysis method. We found that 4-(piperidine-1-sulfonyl)-benzoic acid had a better EC50 than probenecid, which is one of the most specific TRPV2 agonists to date. Exploring the TRPV2-dependent anti-hypertensive potential in vivo, we found that 4-(piperidine-1-sulfonyl)-benzoic acid shows a sex-biased vasodilator effect producing larger vascular relaxations in female mice. Overall, this study expands the pharmacological toolbox for TRPV2, a widely expressed membrane protein and orphan drug target.
RESUMEN
The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to ß-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to ß-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin.
Asunto(s)
Protones , Rodopsina , Temperatura , Rodopsina/genética , Rodopsina/química , Bases de Schiff/química , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Mid-infrared spectroscopy probes molecular vibrations to identify chemical species and functional groups. Therefore, mid-infrared hyperspectral imaging is one of the most powerful and promising candidates for chemical imaging using optical methods. Yet high-speed and entire bandwidth mid-infrared hyperspectral imaging has not been realized. Here we report a mid-infrared hyperspectral chemical imaging technique that uses chirped pulse upconversion of sub-cycle pulses at the image plane. This technique offers a lateral resolution of 15 µm, and the field of view is adjustable between 800 µm × 600 µm to 12 mm × 9 mm. The hyperspectral imaging produces a 640 × 480 pixel image in 8 s, which covers a spectral range of 640-3015 cm-1, comprising 1069 wavelength points and offering a wavenumber resolution of 2.6-3.7 cm-1. For discrete frequency mid-infrared imaging, the measurement speed reaches a frame rate of 5 kHz, the repetition rate of the laser. As a demonstration, we effectively identified and mapped different components in a microfluidic device, plant cell, and mouse embryo section. The great capacity and latent force of this technique in chemical imaging promise to be applied to many fields such as chemical analysis, biology, and medicine.