RESUMO
A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet-visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid CâO stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 â Gln and Ser97 â Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 â Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.
Assuntos
Temperatura Baixa , Rodopsina/química , Substituição de Aminoácidos , Ânions , Etilenos/química , Pichia/química , Conformação Proteica , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral RamanRESUMO
Optogenetics relies on the expression of specific microbial rhodopsins in the neuronal plasma membrane. Most notably, this includes channelrhodopsins, which when heterologously expressed in neurons function as light-gated cation channels. Recently, a new class of microbial rhodopsins, termed anion channel rhodopsins (ACRs), has been discovered. These proteins function as efficient light-activated channels strictly selective for anions. They exclude the flow of protons and other cations and cause hyperpolarization of the membrane potential in neurons by allowing the inward flow of chloride ions. In this study, confocal near-infrared resonance Raman spectroscopy (RRS) along with hydrogen/deuterium exchange, retinal analogue substitution, and site-directed mutagenesis were used to study the retinal structure as well as its interactions with the protein in the unphotolyzed state of an ACR from Guillardia theta (GtACR1). These measurements reveal that (i) the retinal chromophore exists as an all-trans configuration with a protonated Schiff base (PSB) very similar to that of bacteriorhodopsin (BR), (ii) the chromophore RRS spectrum is insensitive to changes in pH from 3 to 11, whereas above this pH the Schiff base (SB) is deprotonated, (iii) when Ser97, the homologue to Asp85 in BR, is replaced with a Glu, it remains in a neutral form (i.e., as a carboxylic acid) but is deprotonated at higher pH to form a blue-shifted species, (iv) Asp234, the homologue of the protonated retinylidene SB counterion Asp212 in BR, does not serve as the primary counteranion for the protonated SB, and (v) substitution of Glu68 with an Gln increases the pH at which SB deprotonation is observed. These results suggest that Glu68 and Asp234 located near the SB exist in a neutral state in unphotolyzed GtACR1 and indicate that other unidentified negative charges stabilize the protonated state of the GtACR1 SB.
Assuntos
Criptófitas/química , Rodopsina/química , Substituição de Aminoácidos , Criptófitas/genética , Conformação Proteica , Retinaldeído/química , Retinaldeído/genética , Retinoides/química , Retinoides/genética , Rodopsina/genética , Bases de Schiff/química , Análise Espectral RamanRESUMO
The filoviruses, Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SEBOV), cause severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Monovalent recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode a filovirus glycoprotein (GP) in place of the VSV glycoprotein, have shown 100% efficacy against homologous filovirus challenge in rodent and NHP studies. Here, we examined the utility of a single-vector, single-injection trivalent rVSV vector expressing MARV, ZEBOV, and SEBOV GPs to protect against MARV-, ZEBOV-, and SEBOV-induced disease in outbred Hartley guinea pigs where we observed protection from effects of all 3 filoviruses.
Assuntos
Infecções por Filoviridae/imunologia , Filoviridae/imunologia , Vetores Genéticos/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Feminino , Infecções por Filoviridae/virologia , Glicoproteínas/imunologia , Cobaias , Vesiculovirus/imunologiaRESUMO
Opsin-based transmembrane voltage sensors (OTVSs) are membrane proteins increasingly used in optogenetic applications to measure voltage changes across cellular membranes. In order to better understand the photophysical properties of OTVSs, we used a combination of UV-Vis absorption, fluorescence and FT-Raman spectroscopy to characterize QuasAr2 and NovArch, two closely related mutants derived from the proton pump archaerhodopsin-3 (AR3). We find both QuasAr2 and NovArch can be optically cycled repeatedly between O-like and M-like states using 5-min exposure to red (660 nm) and near-UV (405 nm) light. Longer red-light exposure resulted in the formation of a long-lived photoproduct similar to pink membrane, previously found to be a photoproduct of the BR O intermediate with a 9-cis retinylidene chromophore configuration. However, unlike QuasAr2 whose O-like state is stable in the dark, NovArch exhibits an O-like state which slowly partially decays in the dark to a stable M-like form with a deprotonated Schiff base and a 13-cis,15-anti retinylidene chromophore configuration. These results reveal a previously unknown complexity in the photochemistry of OTVSs including the ability to optically switch between different long-lived states. The possible molecular basis of these newly discovered properties along with potential optogenetic and biotechnological applications are discussed.
Assuntos
Bacteriorodopsinas , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Opsinas/metabolismo , Fotoquímica , Bombas de Próton , Análise Espectral RamanRESUMO
Many applications in pharmaceutical development, clinical diagnostics, and biological research demand rapid detection of multiple analytes (multiplexed detection) in a minimal volume. This need has led to the development of several novel array-based sensors. The most successful of these so far have been suspension arrays based on polystyrene beads. However, the 5 microm beads used for these assays are incompatible with most microfluidic chip technologies, mostly due to clogging problems. The challenge, then, is to design a detection particle that has high information content (for multiplexed detection), is compatible with miniaturization, and can be manufactured easily at low cost. DNA is a solid molecular wire that is easily produced and manipulated, which makes it a useful material for nanoparticles. DNA molecules are very information-rich, readily deformable, and easily propagated. We exploit these attributes in a suspension array sensor built from specialized recombinant DNA, Digital DNA, that carries both specific analyte-recognition units, and a geometrically encoded identification pattern. Here we show that this sensor combines high multiplexing with high sensitivity, is biocompatible, and has sufficiently small particle size to be used within microfluidic chips that are only 1 microm deep. We expect this technology will be the foundation of a broadly applicable technique to identify and quantitate proteins, nucleic acids, viruses, and toxins simultaneously in a minimal volume.
Assuntos
DNA/análise , Microfluídica/métodos , Proteínas/análise , ImunoensaioRESUMO
Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT-Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all-trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single-/double-bond system similar to the O-intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13-cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR-OTVSs, especially for applications such as deep brain imaging.
Assuntos
Proteínas de Membrana/química , Retinaldeído/química , Análise Espectral/métodos , Sequência de Aminoácidos , OptogenéticaRESUMO
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/métodosRESUMO
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.