A Schiff base connectivity switch in sensory rhodopsin signaling.

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor.

In haloarchaea, light-driven ion transporters have been modified by evolution to produce sensory receptors that relay light signals to transducer proteins controlling motility behavior. The proton pump bacteriorhodopsin and the phototaxis receptor sensory rhodopsin II (SRII) differ by 74% of their residues, with nearly all conserved residues within the photoreactive retinal-binding pocket in the membrane-embedded center of the proteins. Here, we show that three residues in bacteriorhodopsin replaced by the corresponding residues in SRII enable bacteriorhodopsin to efficiently relay the retinal photoisomerization signal to the SRII integral membrane transducer (HtrII) and induce robust phototaxis responses. A single replacement (Ala-215-Thr), bridging the retinal and the membrane-embedded surface, confers weak phototaxis signaling activity, and the additional two (surface substitutions Pro-200-Thr and Val-210-Tyr), expected to align bacteriorhodopsin and HtrII in similar juxtaposition as SRII and HtrII, greatly enhance the signaling. In SRII, the three residues form a chain of hydrogen bonds from the retinal's photoisomerized C(13)=C(14) double bond to residues in the membrane-embedded alpha-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRII's hydrogen bond between Tyr-174, which is in contact with the retinal, and Thr-204, which borders residues on the SRII surface in contact with HtrII, followed by transfer of this chemical energy to drive structural transitions in the transducer helices. The results demonstrate that evolution accomplished an elegant but simple conversion: The essential differences between transport and signaling proteins in the rhodopsin family are far less than previously imagined.

Laser-induced transient grating analysis of dynamics of interaction between sensory rhodopsin II D75N and the HtrII transducer.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (DeltaHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K(1)-K(4)) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the DeltaHtrII portion was found to cause a DeltaHtrII-dependent increase in volume rising in 8 micros in the K(4) state and a drastic decrease in the diffusion coefficient (D) of K(4) relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of DeltaHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Homology Cloning, Heterologous Expression and Characterization of a New Channelrhodopsin

Channelrhodopsins are phototaxis receptors in the plasma membranes of motile unicellular algae. They function as light-gated cation channels and this channel activity has been exploited to trigger action potentials in neurons with light to control neural circuits (“optogenetics"). Four channelrhodopsins were identified in two algal species, Chlamydomonas reinhardtii and Volvox carteri, with known genome sequences; each species contains 2 channelrhodopsins, one absorbing at longer wavelengths and one at shorter wavelengths, named CrChR1 and CrChR2, respectively. Our goals are to expand knowledge of channelrhodopsin mechanisms and also to identify new channelrhodopsins from various algal species with improved properties for optogenetic use. For these aims we are targeting algae from extreme environments to establish the natural diversity of their properties. We cloned a new channelrhodopsin from the psychrophilic (cold-loving) alga, Chlamydomonas augustae, with degenerate primers based on the 4 known homologs. The new protein is 48% and 52% identical to CrChR1 and CrChR2, respectively. We expressed the channelrhodopsin in HEK293 cells and measured light-induced currents to assess their kinetics and action spectrum. Based on the primary structure, kinetics of light-induced photocurrents in HEK293 cells, and action spectrum maximum of 520 nm near that of the two previously found CrChR1, we named the new channelrhodopsin CaChR1. The properties of robust channel activity at physiological pH, fast on-and-off kinetics, and greatly red-shifted action spectrum maximum from that of CrChR2, make CaChR1 advantageous as an optogenetic tool. To know this new channelrhodopsin better, we expressed His-tagged CaChR1 in Pichia pastoris and the yield is about 6 mg/L. The purified His-tagged CaChR1 exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The future work will be measurement of the photocycles of CaChR1 by flash photolysis, crystallization of CaChR1 for the structure and mutagenesis of CaChR1 to find the critical amino acids accounting for red-shifted spectra, slow inactivation and rapid on-and-off kinetics. Seven new channelrhodopsins including CaChR1 from different algal species have been cloned in our lab at this time, bringing the total known to 13. The work of cloning of these new channelrhodopsins along with the expression of CaChR1 was published in Photochemistry and Photobiology in January 2012

Effects of elevated CO2 on early life history development of the yellowtail kingfish, Seriola lalandi, a large pelagic fish

An increasing number of studies have examined the effects of elevated carbon dioxide (CO2) and ocean acidification on marine fish, yet little is known about the effects on large pelagic fish. We tested the effects of elevated CO2 on the early life history development and behaviour of yellowtail kingfish, Seriola lalandi. Eggs and larvae were reared in current day control (450 µatm) and two elevated CO2 treatments for a total of 6 d, from 12 h post-fertilization until 3 d post-hatching (dph). Elevated CO2 treatments matched projections for the open ocean by the year 2100 under RCP 8.5 (880 µatm CO2) and a higher level (1700 µatm CO2) relevant to upwelling zones where pelagic fish often spawn. There was no effect of elevated CO2 on survival to hatching or 3 dph. Oil globule diameter decreased with an increasing CO2 level, indicating potential effects of elevated CO2 on energy utilization of newly hatched larvae, but other morphometric traits did not differ among treatments. Contrary to expectations, there were no effects of elevated CO2 on larval behaviour. Activity level, startle response, and phototaxis did not differ among treatments. Our results contrast with findings for reef fish, where a wide range of sensory and behavioural effects have been reported. We hypothesize that the absence of behavioural effects in 3 dph yellowtail kingfish is due to the early developmental state of newly hatched pelagic fish. Behavioural effects of high CO2 may not occur until larvae commence branchial acid-base regulation when the gills develop; however, further studies are required to test this hypothesis. Our results suggest that the early stages of kingfish development are tolerant to rising CO2 levels in the ocean.

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and/or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor/transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum, which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. These latter results may be exploited to further analyze the binding interaction of the photoreceptors with their downstream effectors.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein.

Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified. On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed. Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII). The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene. The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm. HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment. The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr. The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments. SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%). The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII. In BR, residues Asp-85 and Asp-96 are crucial in proton pumping. In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr. Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions. This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

Measurement of action spectra of light-activated processes

We report on a new experimental technique suitable for measurement of light-activated processes, such as fluorophore transport. The usefulness of this technique is derived from its capacity to decouple the imaging and activation processes, allowing fluorescent imaging of fluorophore transport at a convenient activation wavelength. We demonstrate the efficiency of this new technique in determination of the action spectrum of the light mediated transport of rhodamine 123 into the parasitic protozoan Giardia duodenalis. (c) 2006 Society of Photo-Optical Instrumentation Engineers.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.