Dependence of locomotion paralysis on light intensity.

<p>Animals expressing Arch::GFP under muscle-specific (<i>myo-3p</i>) and pan-neuronal (<i>aex-3p</i> and <i>F25B3.3p</i>) promoters were illuminated with green light at varying intensities. Animals with <i>nc3034Ex[F25B3.3p::Arch::gfp]</i> (squares) and <i>nc3026Ex[aex-3p::Arch::gfp]</i> (diamonds) exhibited higher responsiveness at lower light intensities than <i>nc3031Ex[myo-3p::Arch::gfp]</i> (triangles) animals did. (mean±SEM; n = 3. Five animals were examined for each trial.).</p

Comparison of light-elicited locomotory paralysis mediated by Arch and Arch::GFP.

<p>(A) Dependence of locomotion paralysis of <i>Ex[hsp-16.2p::Arch]</i>animals (black squares) and <i>Ex[hsp-16.2p::Arch::gfp]</i> animals (open circles) to light intensity was measured 12h after heat shock. (B) Time course of light-elicited locomotory paralysis after heat-shock induction of Arch and Arch::GFP. Heat-shocked <i>Ex[hsp-16.2p::Arch]</i> (black squares) and <i>Ex hsp-16.2p::Arch::gfp]</i> (open circles) animals were examined at each time point as shown in Fig. 4. A(i). Three independently isolated lines were used for each transgene: <i>ncEx3002, ncEx3003</i> and <i>ncEx3004</i> for <i>Ex[hsp-16.2p::Arch::gfp]</i>, and <i>ncEx3039, ncEx3040</i> and <i>ncEx3041</i> for <i>Ex[hsp16-2p::Arch]</i>. Shown are the mean±SEM of 9 trials consisting of 3 trials for each line. Five animals were examined for each trial.</p

Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation

Assembly is one of the keys to understand biological molecules, and it takes place in spatial and temporal domains upon stimulation. Microbial rhodopsin (also called retinal protein) is a membrane-embedded protein that has a retinal chromophore within seven-transmembrane α-helices and shows homo-, di-, tri-, penta-, and hexameric assemblies. Those assemblies are closely related to critical physiological properties such as stabilizing the protein structure and regulating their photoreaction dynamics. Here we investigated the assembly and disassembly of thermophilic rhodopsin (TR), which is a novel proton-pumping rhodopsin derived from a thermophile living at 75 °C. TR was characterized using size-exclusion chromatography and circular dichroism spectroscopy, and formed a trimer at 25 °C, but irreversibly dissociated into monomers upon thermal stimulation. The transition temperature was estimated to be 68 °C. The irreversible nature made it possible to investigate the photochemical properties of both the trimer and the monomer independently. Compared with the trimer, the absorption maximum of the monomer is blue-shifted by 6 nm without any changes in the retinal composition, p<i>K</i>_a value for the counterion or the sequence of the proton movement. The photocycling rate of the monomeric TR was similar to that of the trimeric TR. A similar trimer-monomer transition upon thermal stimulation was observed for another eubacterial rhodopsin GR but not for the archaeal rhodopsins AR3 and HwBR, suggesting that the transition is conserved in bacterial rhodopsins. Thus, the thermal stimulation of TR induces the irreversible disassembly of the trimer

Locomotion assay using heat shock-mediated induction of Arch.

<p>(A) Scheme showing the schedule of transferring worms to plates with or without all-trans-retinal (ATR) in heat shock induction experiments. i: Animals were cultivated in the presence of ATR throughout the experiments. (Figs. 3B, 4B) ii: Animals were transferred to ATR-supplemented plates 24 h after heat shock (Fig. 4B). iii: Animals were cultivated in the presence of ATR and transferred to ATR-free plates 24 h after heat shock. (B) Time course of light-elicited locomotory paralysis after heat-shock induction of Arch::GFP. Heat-shocked <i>nc3003Ex[hsp-16.2p::Arch::gfp]</i> animals cultivated in the presence of ATR throughout the experiment (circles) (Fig. 4. A(i)) or transferred from ATR-free to ATR-supplemented plates 24 h after heat shock (triangles) (Fig. 4. A(ii)) were examined at each time point. When ATR was present throughout the experiment (circles), paralysis of worms was first noticed 6 h after heat shock. The paralysis rate reached a plateau 12 h after heat shock, and remained constant for 48 h. (mean±SEM; n = 3, Five animals were examined for each trial.) Worms cultivated in the absence of ATR throughout the experiment did not respond to illumination at any time point. When animals were grown and heat shocked in the absence of ATR and then transferred to ATR-supplemented plates 24 h later (triangles), half of them were paralyzed by illumination 1.5 h after transfer, and the paralysis rate reached a plateau within 3 h. (mean±SEM; n = 4, Five animals were examined for each trial.).</p

Defects in the locomotory behavior caused by silencing subsets of motor neurons.

<p>(A) A crawling track of an <i>ncEx2351[unc-47p::Arch::gfp]</i> animal. When the freely moving animal was illuminated with green light for 1 minute, it performed loopy movement (dotted square). After turning off the green light, it resumed normal sinusoidal movement. Scale bar = 500 µm (B-D) Locomotory behavior of worms expressing Arch in motor neurons subsets. Arch::GFP was expressed in D-type (VD, DD), A-type (VA, DA), B-type (VB, DB) and VA +VB motor neurons in <i>ncEx2351[unc-47p::Arch::gfp], ncEx2371[acr-5p::Arch::gfp], ncEx3068[unc-4p::Arch::gfp]</i> and <i>ncEx2365[del-1p::Arch::gfp]</i> animals, respectively. Locomotion of <i>unc-47(e307)</i> animals was also scored. For all transgenic strains, animals behaviors under green light illumination (ON = open box) and those without illumination (OFF = filled box) differed significantly (p<0.001, Fisher's exact test for the locomotion assay under the freely moving condition and Student's <i>t</i> test for the touch response assay). Exceptions are forward movement of <i>ncEx3068[unc-4p::Arch::gfp]</i> animals, which did not change significantly under illumination, and forward movement of <i>ncEx2365[del-1p::Arch::gfp]</i> animals elicited by gentle posterior touch (p = 0.012). Error bars indicate ±SEM. (B) Forward (F) and backward movement (B) was scored in worms moving freely. Percentage of animals exhibiting the “Class 3 (severe)”, “Class2 (mild)” and “Class1 (no)” phenotype in locomotory behaviors when they were illuminated with green light is shown. Defects were classified as “Class 3″ when no corresponding movement or response was observed. Other abnormalities, such as retardation or decrease in the extent of movement, are classified as “Class2”. (C) Forward movement (F) to gentle posterior touch and backward (B) movement to gentle anterior touch were scored. Responses out of five touches are shown. (D) Forward (F) and backward movement (B) to harsh touch was scored, and was shown additively in each bar. Responses out of five touches are shown.</p

Expression of Arch::GFP in <i>C. elegans</i> driven by various promoters.

<p>(A) A fluorescent micrograph of an <i>nc3031Ex[myo-3p::Arch::gfp]</i> animal. Arch::GFP is expressed in longitudinal bands composed of body wall muscles (arrow). (B) An enlarged view of body wall muscle cells. GFP signal is observed along the outline of muscle cells (arrow). Vesicular structures visualized with GFP are localized close to the cell membrane (arrow head). Weak GFP signal is detected in the cytoplasm (asterisk). (C) Expression of Arch::GFP in an <i>nc3034Ex[F25B3.3p::Arch::gfp]</i> animal. Arch::GFP is expressed in head neurons (arrow head), tail neurons (open arrow head) and the ventral nerve cord (arrow). (D) The head of an animal carrying <i>F25B3.3p::Arch::gfp</i>. Arch::GFP is expressed in the axon (arrow) and the cell body (arrow head) of head neurons. (E) Expression of Arch::GFP in an <i>nc3026Ex[aex-3p::Arch::gfp]</i> animal. Arch::GFP is expressed in head neurons (arrow), tail neurons (arrow head), and the ventral nerve cord (open arrow head). (F) The head of an animal carrying <i>aex-3p::Arch::gfp</i>. Arch::GFP is expressed in the axon of head neurons in a punctured pattern (arrow). GFP is seen on the cell membrane of a cell body (arrow head). (G) Expression of Arch::GFP in an <i>nc3003Ex[hsp-16.2p::Arch::gfp]</i> animal. Arch::GFP is expressed everywhere in the body. (H) The head of an animal carrying <i>hsp-16.2p:: Arch::gfp</i>. Arch::GFP is expressed in neurons (arrow). (I) Body wall muscle of an <i>hsp-16.2p::Arch::gfp</i> animal. GFP is clearly localized at the cell membrane (arrow). Scale bar: A, C, E, G = 100 µm; B, D, F, H I = 10 µm. Anterior is toward the right except for (G).</p

Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics

Several new retinal-based photoreceptor proteins that act as light-driven electrogenic halide ion pumps have recently been discovered. Some of them, called “NTQ” rhodopsins, contain a conserved Asn–Thr–Gln motif in the third or C-helix. In this study, we investigated the photochemical characteristics of an NTQ rhodopsin, Nonlabens marinus rhodopsin 3 (NM-R3), which was discovered in the N. marinus S1-08^T strain, using static and time-resolved spectroscopic techniques. We demonstrate that NM-R3 binds a Cl^– in the vicinity of the retinal chromophore accompanied by a spectral blueshift from 568 nm in the absence of Cl^– to 534 nm in the presence of Cl^–. From the Cl^– concentration dependence, we estimated the affinity (dissociation constant, <i>K</i>_d) for Cl^– in the original state as 24 mM, which is ca. 10 times weaker than that of archaeal halorhodopsins but ca. 3 times stronger than that of a marine bacterial Cl^– pumping rhodopsin (C1R). NM-R3 showed no dark–light adaptation of the retinal chromophore and predominantly possessed an all-<i>trans</i>-retinal, which is responsible for the light-driven Cl^– pump function. Flash-photolysis experiments suggest that NM-R3 passes through five or six photochemically distinct intermediates (K, L­(N), O₁, O₂, and NM-R3′). From these results, we assume that the Cl^– is released and taken up during the L­(N)–O₁ transition from a transiently formed cytoplasmic (CP) binding site and the O₂–NM-R3′ or the NM-R3′–original NM-R3 transitions from the extracellular (EC) side, respectively. We propose a mechanism for the Cl^– transport by NM-R3 based on our results and its recently reported crystal structure

The Early Steps in the Photocycle of a Photosensor Protein Sensory Rhodopsin I from Salinibacter ruber

Light absorption by the photoreceptor microbial rhodopsin triggers <i>trans</i>–<i>cis</i> isomerization of the retinal chromophore surrounded by seven transmembrane α-helices. Sensory rhodopsin I (SRI) is a dual functional photosensory rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (<i>Sr</i>SRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the S_n←S₁ absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial rhodopsins. The first dynamics (τ₁ ∼ 54 fs) corresponds to the population branching process from the Franck<b>–</b>Condon region to the reactive (S₁^r) and nonreactive (S₁^nr) S₁ states. The second dynamics (τ₂ = 0.64 ps) is the isomerization process of the S₁^r state to generate the ground-state 13-<i>cis</i> form, and the third dynamics (τ₃ = 1.8 ps) corresponds to the internal conversion of the S₁^nr state. The fourth component (τ₃′ = 2.5 ps) is assignable to the J-decay (K-formation). This reaction scheme was further supported by the results of fluorescence spectroscopy. To investigate the protein response(s), the spectral changes of the tryptophan bands were monitored by ultraviolet resonance Raman spectroscopy. The intensity change following the K formation in the chromophore structure (τ ∼ 17 ps) was significantly small in <i>Sr</i>SRI as compared with other microbial rhodopsins. We also analyzed the effect(s) of Cl^– binding on the ultrafast dynamics of <i>Sr</i>SRI. Compared with a chloride pump Halorhodopsin, Cl^– binding to <i>Sr</i>SRI was less effective for the excited-state dynamics, whereas the binding altered the structural changes of tryptophan following the K-formation, which was the characteristic feature for <i>Sr</i>SRI. On the basis of these results, a primary photoreaction scheme of <i>Sr</i>SRI together with the role of chloride binding is proposed

Influence of Halide Binding on the Hydrogen Bonding Network in the Active Site of <i>Salinibacter</i> Sensory Rhodopsin I

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon <i>Halobacterium salinarum</i>. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from <i>Salinibacter ruber</i> (<i>Sr</i>SRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) <i>J. Mol. Biol.</i> <i>392</i>, 48–62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in <i>Sr</i>SRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties

Temperature Independence of Ultrafast Photoisomerization in Thermophilic Rhodopsin: Assessment versus Other Microbial Proton Pumps

Primary photochemical events in the unusually thermostable proton pumping rhodopsin of <i>Thermus thermophilus</i> bacterium (TR) are reported for the first time. Internal conversion in this protein is shown to be significantly faster than in bacteriorhodopsin (BR), making it the most rapidly isomerizing microbial proton pump known. Internal conversion (IC) dynamics of TR and BR were recorded from room temperature to the verge of thermal denaturation at 70 °C and found to be totally independent of temperature in this range. This included the well documented multiexponential nature of IC in BR, suggesting that assignment of this to ground state structural inhomogeneity needs revision. TR photodynamics were also compared with that of the phylogenetically more similar proton pump <i>Gloeobacter</i> rhodopsin (GR). Despite this similarity GR has poor thermal stability, and the excited state decays significantly more slowly and exhibits very prominent stretched exponential behavior. Coherent torsional wave-packets induced by impulsive photoexcitation of TR and GR show marked resemblance to each other in frequency and amplitude and differ strikingly from similar signatures in pump–probe data of BR and other microbial retinal proteins. Possible correlations between IC rates and thermal stability and the promise of using torsional coherence signatures for understanding chromophore protein binding in microbial retinal proteins are discussed