Sensory Rhodopsin Research Articles

Molecular modeling of the HAMP domain of sensory rhodopsin II transducer from Natronomonas pharaonis.

The halobacterial transducer of sensory rhodopsin II (HtrII) is a photosignal transducer associated with phototaxis in extreme halophiles. The HAMP domain, a linker domain in HtrII, is considered to play an important role in transferring the signal from the membrane to the cytoplasmic region, although its structure in the complex remains undetermined. To establish the structural basis for understanding the mechanism of signal transduction, we present an atomic model of the structure of the N-terminal HAMP domain from Natronomonas pharaonis (HtrII: 84–136), based on molecular dynamics (MD) simulations. The model was built by homology modeling using the NMR structure of Af1503 from Archaeoglobus fulgidus as a template. The HAMP domains of Af1503 and HtrII were stable during MD simulations over 100 ns. Quantitative analyses of inter-helical packing indicated that the Af1503 HAMP domain stably maintained unusual knobs-to-knobs packing, as observed in the NMR structure, while the bulky side-chains of HtrII shifted the packing state to canonical knobs-into-holes. The role of the connector loop in maintaining structural stability was also discussed using MD simulations of loop deletion mutants.

1P259 大腸菌における光誘導型タンパク質発現制御系の確立に向けて(光生物-視覚・光受容,第48回日本生物物理学会年会)

1P259 大腸菌における光誘導型タンパク質発現制御系の確立に向けて(光生物-視覚・光受容,第48回日本生物物理学会年会)

2P116 Three-dimensional Solid-state NMR study of Anabaena Sensory Rhodopsin in the lipid environment : Chemical Shift Assignments(The 48th Annual Meeting of the Biophysical Society of Japan)

2P116 Three-dimensional Solid-state NMR study of Anabaena Sensory Rhodopsin in the lipid environment : Chemical Shift Assignments(The 48th Annual Meeting of the Biophysical Society of Japan)

Photoreactions and Structural Changes of Anabaena Sensory Rhodopsin

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) which is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. This article reviews the detailed photoreaction processes of ASR, which were studied by low-temperature Fourier-transform infrared (FTIR) and UV-visible spectroscopy. The former research reveals that the retinal isomerization is similar to bacteriorhodopsin (BR), but the hydrogen-bonding network around the Schiff base and cytoplasmic region is different. The latter study shows the stable photoproduct of the all-trans form is 100% 13-cis, and that of the 13-cis form is 100% all-trans. These results suggest that the structural changes of ASR in the cytoplasmic domain play important roles in the activation of the transducer protein, and photochromic reaction is optimized for its sensor function.

Heterogeneity in the Nitroxide Micro-Environment: Polarity and Proticity Effects in Spin-Labeled Proteins Studied by Multi-Frequency EPR

This study aims to investigate the g xx heterogeneity of the g-tensor commonly observed in high-field electron paramagnetic resonance (EPR) spectra of nitroxide spin-labeled sites in proteins. This heterogeneity is addressed in terms of spin-label populations characterized by different polarity and H-bonding properties of the nitroxide micro-environment. The g xx value for each population is determined from the fit of continuous-wave high-field spectra obtained at 95, 275 and 360 GHz with a series of nitroxide spin-labels covalently attached to different sites in both membrane and water-soluble proteins. The spin-labeled proteins investigated include sensory rhodopsin II and its cognate transducer molecule (HtrII) from Natronomonas pharaonis both in micelles and membranes, bacteriorhodopsin from Halobacterium salinarum in native purple membrane lipid bilayers and water-soluble colicin A from Escherichia coli. To avoid contributions to the g xx spectral features of the nitroxide label due to nuclear quadrupole interactions arising from 14N nuclei, and to simplify the nitrogen hyperfine pattern, methanethiosulfonate spin labels, containing the 15N isotope (I = 1/2) in some experiments, were employed. A consistent analysis of all multi-frequency EPR spectra revealed three distinct g xx values, g i xx , for each investigated position of the labeled proteins. In contrast, distinctly different nitrogen hyperfine splittings A zz of the nitroxides in the various labeled proteins could not be resolved, but rather an average hyperfine splitting \(\bar{A}_{{zz}}\) was obtained. The g i xx values as well as the fractions of the different nitroxide populations were found to be correlated with the average hyperfine constant \(\bar{A}_{{zz}},\) a parameter which likewise is known to be sensitive to the local polarity of the spin-label micro-environment. Plotting the different g i xx values obtained for each EPR spectrum versus \(\bar{A}_{{zz}}\) of the labeled proteins reveals new interesting aspects of the nitroxide label micro-environment in terms of polarity and H-bonding propensity (proticity). Linear approximations of the different regions of the plot g i xx versus \(\bar{A}_{{zz}}\) are presented and compared with theoretical and experimental data available from the literature.

Engineering an Inward Proton Transport from a Bacterial Sensor Rhodopsin

ATP is synthesized by an enzyme that utilizes proton motive force, and thus, nature has created various proton pumps. The best-understood proton pump is bacteriorhodopsin (BR), an outward-directed, light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. We previously converted BR into an inward-directed chloride ion pump, but an inward proton pump has never been created. Proton pumps must have a specific mechanism to exclude transport in the reverse direction in order to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Here we report that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement. Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria that possesses little proton pump activity. When we replaced Asp217 in the cytoplasmic domain (a distance of approximately 15 A from the retinal chromophore) by Glu, ASR exhibited an inward proton transport activity driven by absorption of a single photon. FTIR spectra clearly showed an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to that in normal proton pumps.

Evolution of phototaxis.

Phototaxis in the broadest sense means positive or negative displacement along a light gradient or vector. Prokaryotes most often use a biased random walk strategy, employing type I sensory rhodopsin photoreceptors and two-component signalling to regulate flagellar reversal. This strategy only allows phototaxis along steep light gradients, as found in microbial mats or sediments. Some filamentous cyanobacteria evolved the ability to steer towards a light vector. Even these cyanobacteria, however, can only navigate in two dimensions, gliding on a surface. In contrast, eukaryotes evolved the capacity to follow a light vector in three dimensions in open water. This strategy requires a polarized organism with a stable form, helical swimming with cilia and a shading or focusing body adjacent to a light sensor to allow for discrimination of light direction. Such arrangement and the ability of three-dimensional phototactic navigation evolved at least eight times independently in eukaryotes. The origin of three-dimensional phototaxis often followed a transition from a benthic to a pelagic lifestyle and the acquisition of chloroplasts either via primary or secondary endosymbiosis. Based on our understanding of the mechanism of phototaxis in single-celled eukaryotes and animal larvae, it is possible to define a series of elementary evolutionary steps, each of potential selective advantage, which can lead to pelagic phototactic navigation. We can conclude that it is relatively easy to evolve phototaxis once cell polarity, ciliary swimming and a stable cell shape are present.

Characterization of a Signaling Complex Composed of Sensory Rhodopsin I and Its Cognate Transducer Protein from the EubacteriumSalinibacter ruber

Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate transducer protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in protein-protein interactions with the kinase CheA and the adaptor protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533-23541]. Here we cloned and expressed SrSRI with its full-length transducer protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 mg/L of culture). In addition, we report here the photochemical properties of the SrSRI-SrHtrI complex using time-resolved laser flash spectroscopy and other spectroscopic techniques and compare them to SrSRI without SrHtrI.

Primary Photoinduced Protein Response in Bacteriorhodopsin and Sensory Rhodopsin II

Essential for the biological function of the light-driven proton pump, bacteriorhodopsin (BR), and the light sensor, sensory rhodopsin II (SRII), is the coupling of the activated retinal chromophore to the hosting protein moiety. In order to explore the dynamics of this process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled BR and SRII samples. These include SRII in D(2)O buffer, BR in H(2)(18)O medium, SRII with (15)N-labeled protein, and BR with (13)C(14)(13)C(15)-labeled retinal chromophore. Via observed shifts of infrared difference bands after photoexcitation and their kinetics we provide evidence for nonchromophore bands in the amide I and the amide II region of BR and SRII. A band around 1550 cm(-1) is very likely due to an amide II vibration. In the amide I region, contributions of modes involving exchangeable protons and modes not involving exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to bending vibrations of protein-bound water molecules. The observed protein bands appear in the amide I region within the system response of ca. 0.3 ps and in the amide II region within 3 ps, and decay partially in both regions on a slower time scale of 9-18 ps. Similar observations have been presented earlier for BR5.12, containing a nonisomerizable chromophore (R. Gross et al. J. Phys. Chem. B 2009, 113, 7851-7860). Thus, the results suggest a common mechanism for ultrafast protein response in the artificial and the native system besides isomerization, which could be induced by initial chromophore polarization.

Primary Reaction of Sensory Rhodopsin II Mutant D75N and the Influence of Azide

The early steps in the photocycle of sensory rhodopsin II mutant D75N are investigated in a comprehensive study using femtosecond visible pump/probe spectroscopy. An overall slower response dynamics after photoexcitation is observed compared to wild-type sensory rhodopsin II, which is assigned to changed electrostatics and an altered hydrogen-bonding network within the retinal binding pocket. Furthermore, the influence of azide on the primary reaction is analyzed. The addition of azide accelerates the sub-10 ps dynamics of the D75N mutant nearly to reaction rates found in wild-type. Moreover, a further reaction pathway becomes observable in the investigated time range, which is assigned to a previously described K(1) to K(2) transition. The specific acceleration of the early steps seems to be a unique feature of the D75N mutant as similar azide effects do not emerge in analogous azide measurements of wild-type sensory rhodopsin II, bacteriorhodopsin, and the bacteriorhodopsin mutant D85N.

The Anabaena sensory rhodopsin transducer defines a novel superfamily of prokaryotic small-molecule binding domains

The Anabaena sensory rhodopsin transducer (ASRT) is a small protein that has been claimed to function as a signaling molecule downstream of the cyanobacterial sensory rhodopsin. However, orthologs of ASRT have been detected in several bacteria that lack rhodopsin, raising questions about the generality of this function. Using sequence profile searches we show that ASRT defines a novel superfamily of β-sandwich fold domains. Through contextual inference based on domain architectures and predicted operons and structural analysis we present strong evidence that these domains bind small molecules, most probably sugars. We propose that the intracellular versions like ASRT probably participate as sensors that regulate a diverse range of sugar metabolism operons or even the light sensory behavior in Anabaena by binding sugars or related metabolites. We also show that one of the extracellular versions define a predicted sugar-binding structure in a novel cell-surface lipoprotein found across actinobacteria, including several pathogens such as Tropheryma, Actinomyces and Thermobifida. The analysis of this superfamily also provides new data to investigate the evolution of carbohydrate binding modes in β-sandwich domains with very different topologies.Reviewers: This article was reviewed by M. Madan Babu and Mark A. Ragan.

Single-Molecule Force Spectroscopy Measures Structural Changes Induced by Light Activation and Transducer Binding in Sensory Rhodopsin II

Microbial rhodopsins are a family of seven-helical transmembrane proteins containing retinal as chromophore. Sensory rhodopsin II (SRII) triggers two very different responses upon light excitation, depending on the presence or the absence of its cognate transducer HtrII: Whereas light activation of the NpSRII/ NpHtrII complex activates a signalling cascade that initiates the photophobic response, NpSRII alone acts as a proton pump. Using single-molecule force spectroscopy, we analysed the stability of NpSRII and its complex with the transducer in the dark and under illumination. By improving force spectroscopic data analysis, we were able to reveal the localisation of occurring forces within the protein chain with a resolution of about six amino acids. Distinct regions in helices G and F were affected differently, depending on the experimental conditions. The results are generally in line with previous data on the molecular stability of NpSRII. Interestingly, new interaction sites were identified upon light activation, whose functional importance is discussed in detail.

Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I

Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum ( HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein ( SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl −-dependent manner with a K m of 307 ± 56 mM, showing that Cl −-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO 3), implying that I −, Br −, and NO 3 − can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl −-binding site. In light of these results, we discuss the effects of the Cl − binding to SRI and the roles of Cl − binding in its function.

Microbial Rhodopsins: Receptors, Channels, and Pumps from a Single Design

The microbial rhodopsin family is comprised of ~4800 homologous proteins containing 7 transmembrane helices forming a pocket for the chromophore retinal. Most are light‐driven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). Since we identified the first sensory rhodopsin in 1982, homologous photosensors have been found to be widely distributed in archaea, eubacteria, and unicellular eukaryotes, and to signal with remarkably diverse mechanisms. Some of the best studied are the phototaxis receptors in haloarchaeal prokaryotes (SRI and SRII) and in eukaryotic algae (channelrhodopsins). SRI and SRII transmit signals by protein‐protein interaction to control a cytoplasmic phosphorylation cascade that modulates the prokaryotic flagellar motors. Mediating algal phototaxis, channelrhodopsins are light‐gated cation channels that depolarize the membrane to trigger Ca++‐influx into the eukaryotic flagellar axoneme. Crystallography, spectroscopy, and genetic engineering have begun to clarify how modifications of the same architecture enable the microbial rhodopsins to carry out their distinctly different molecular functions. Interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions.

Protein Kinase A Activity Controls the Regulation of T-type CaV3.2 Channels by Gβγ Dimers

Low voltage-activated (LVA), T-type, calcium channels mediate diverse biological functions and are inhibited by Gbetagamma dimers, yet the molecular events required for channel inhibition remain unknown. Here, we identify protein kinase A (PKA) as a molecular switch that allows Gbeta(2)gammax dimers to effect voltage-independent inhibition of Ca(v)3.2 channels. Inhibition requires phosphorylation of Ser(1107), a critical serine residue on the II-III loop of the channel pore protein. S1107A prevents inhibition of unitary currents by recombinant Gbeta(2)gamma(2) dimers but does not disrupt dimer binding nor change its specificity. Gbetagamma dimers released upon receptor activation also require PKA activity for their inhibitory actions. Hence, dopamine inhibition of Ca(v)3.2 whole cell current is precluded by Gbetagamma-scavenger proteins or a peptide that blocks PKA catalytic activity. Fittingly, when used alone at receptor-selective concentrations, D(1) or D(2) agonists do not elicit channel inhibition yet together synergize to inhibit Ca(v)3.2 channel currents. We propose that a dual-receptor regulatory mechanism is used by dopamine to control Ca(v)3.2 channel activity. This mechanism, for example, would be important in aldosterone producing adrenal glomerulosa cells where channel dysregulation would lead to overproduction of aldosterone and consequent cardiac, renal, and brain target organ damage.

Photosensory Functions of Channelrhodopsins in Native Algal Cells†

Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light-gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin-2) is biochemical activation of secondary Ca(2+)-channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin-1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low-efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells.

Functional Expression of the Signaling Complex Sensory Rhodopsin II/Transducer II from Halobacterium salinarum in Escherichia coli†

Sensory rhodopsin II, a photoreceptor from Halobacterium salinarum (HsSRII), in complex with its cognate transducer protein (HsHtrII) triggers the photophobic response via a cytoplasmic two-component signaling cascade. HsHtrII possess in addition to the HsSRII binding and the cytoplasmic domains an extracellular serine-receptor domain. Here we describe the properties of HsSRII and HsHtrII and those of various shortened transducer analogs, heterologously expressed in Escherichia coli. HsSRII displays the photocycle typical of archaeal photosensors with prolonged kinetics. Using an isothermal titration calorimetric analysis for this complex a dissociation constant of 1.1 microm was obtained similar to that of the corresponding transducer/receptor pair from Natronobacterium pharaonis. A shortened transducer lacking the extracellular and cytoplasmic domain is also sufficient to bind the receptor with a slightly lower affinity. The dissociation constant of serine binding to the extracellular domain was determined to be about 5 microm. This result is in line with the proposal that the extracellular domain indeed is a serine receptor.

Vectorial Ion Transport by Channelrhodopsin-2

Channelrhodopsins represent a third member of microbial-type rhodopsins and have gained considerable attention in neurobiology as a tool to control the excitability of neurons. The other photoreceptors act either as light-driven ion pumps like bacteriorhodopsin (BR) or as part of a relay system like the sensory rhodopsins (SR). In contrast, channelrhodopsins are light-gated ion channels that allow the passive permeation of cations over the membrane barrier after light activation. Previously, we could follow the spectral characteristics of different photointermediates and relate them to the functional states of the channel. In the gating process, the Schiff-base undergoes a deprotonation reaction before it relaxes into a red-shifted species. The red-shifted photointermediate is characteristic for the open state with a lifetime of 10 ms.So far, the role of the protonation reactions is not fully understood. Channelrhodopsins possess a glutamate residue at the homologue position of the proton acceptor D85 in BR. The main difference among the microbial-type rhodopsins is found in the nature of the proton donor at the opposite side of the retinal moiety towards the cytoplasm: In BR, an aspartic acid reprotonates the Schiff-base in a fast manner as expected for an efficient proton pump, while in SR an aromatic residue leads to a long-lived deprotonated state of the Schiff-base. In channelrhodopsins, the homologue residue is a histidine that could allow a reprotonation of the observed deprotonated Schiff-base from the cytoplasmic side. As a net result, one proton would be transported per photocycle. Here we show that one can indeed observe such a vectorial ion translocation under illumination of purified and proteoliposome reconstituted channelrhodopsin-2. Therefore, the mechanism of channelrhodopsin-2 shows similar features as other rhodopsins, i.e. control of the accessibility to the Schiff-base by light-induced isomerization of the retinal and vectorial ion transport.

Atomic Force Spectroscopy Measures Light Activation And Transducer Binding Induced Structural Changes In The Sensory Rhodopsin II

Rhodopsins are a family of seven-helical transmembrane proteins responding to light. Sensory rhodopsin II (SRII) triggers two very different responses depending on the presence or absence of its transducer: Whereas light activation of the SRII-transducer-complex triggers a signalling cascade initiating the photophobic response of the bacterium, SRII alone acts as a proton-pump.Using single molecule force spectroscopy we analysed the stability of SRII in dark and after light activation as well as in presence and absence of the transducer, which revealed a distinct pattern of changes in the protein stability. By improving the force spectroscopic data analysis we were able to predict the localisation of occuring forces within the protein chain with a resolution of about six amino acids.Different regions showed up, where secondary structure elements of SRII are selectively stabilised or weakened by either light activation or transducer binding or both. Independent of the presence of the transducer light activation has a destabilizing effect in the middle of α-helix G. This suggests a loss in interactions between helices G and F, which would allow an outward tilt of α-helix F as previously observed. Additionally, the unfolding curves show an increased number of rupture events in the region of helix F upon transducer binding, which is most likely due to the formation of several interactions between α-helix F and TM2 of the transducer. Most interestingly, we found a loss of some of these interactions upon light activation, which might explain transducer activation and help to answer remaining questions concerning the precise molecular mechanism. Finally, in the absence of the transducer, destabilizing effects are observed at the cytoplasmic half of helix G, which might indicate its importance for the proton-pumping properties of SRII.

Active Water in Protein−Protein Communication within the Membrane: The Case of SRII−HtrII Signal Relay

We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.