John L. Spudich

Proteorhodopsin phototrophy in the ocean

Proteorhodopsin, a retinal-containing integral membrane protein that functions as a light-driven proton pump, was discovered in the genome of an uncultivated marine bacterium; however, the prevalence, expression and genetic variability of this protein in native marine microbial populations remain unknown. Here we report that photoactive proteorhodopsin is present in oceanic surface waters. We also provide evidence of an extensive family of globally distributed proteorhodopsin variants. The protein pigments comprising this rhodopsin family seem to be spectrally tuned to different habitats—absorbing light at different wavelengths in accordance with light available in the environment. Together, our data suggest that proteorhodopsin-based phototrophy is a globally significant oceanic microbial process.

Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii

We demonstrate that two rhodopsins, identified from cDNA sequences, function as low- and high-light-intensity phototaxis receptors in the eukaryotic alga Chlamydomonas reinhardtii. Each of the receptors consists of an ≈300-residue seven-transmembrane helix domain with a retinal-binding pocket homologous to that of archaeal rhodopsins, followed by ≈400 residues of additional membrane-associated portion. The function of the two rhodopsins, Chlamydomonas sensory rhodopsins A and B (CSRA and CSRB), as phototaxis receptors is demonstrated by in vivo analysis of photoreceptor electrical currents and motility responses in transformants with RNA interference (RNAi) directed against each of the rhodopsin genes. The kinetics, fluence dependencies, and action spectra of the photoreceptor currents differ greatly in transformants in accord with the relative amounts of photoreceptor pigments expressed. The data show that CSRA has an absorption maximum near 510 nm and mediates a fast photoreceptor current that saturates at high light intensity. In contrast, CSRB absorbs maximally at 470 nm and generates a slow photoreceptor current saturating at low light intensity. The relative wavelength dependence of CSRA and CSRB activity in producing phototaxis responses matches precisely the wavelength dependence of the CSRA- and CSRB-generated currents, demonstrating that each receptor mediates phototaxis. The saturation of the two photoreceptor currents at different light fluence levels extends the range of light intensity to which the organism can respond. Further, at intensities where both operate, their light signals are integrated at the level of membrane depolarization caused by the two photoreceptor currents.

Diversification and spectral tuning in marine proteorhodopsins

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently discovered in the cosmopolitan uncultured SAR86 bacterial group in oceanic surface waters. Two related proteorhodopsin families were found that absorb light with different absorption maxima, 525 nm (green) and 490 nm (blue), and their distribution was shown to be stratified with depth. Using structural modeling comparisons and mutagenesis, we report here on a single amino acid residue at position 105 that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families. Furthermore, looking at natural environments, we found novel proteorhodopsin gene clusters spanning the range of 540–505 nm and containing changes in the same identified key switch residue leading to changes in their absorption maxima. The results suggest a simultaneous diversification of green proteorhodopsin and the new key switch variant pigments. Our observations demonstrate that this single‐residue switch mechanism is the major determinant of proteorhodopsin wavelength regulation in natural marine environments.

Handbook of Photosensory Receptors

Preface. List of Authors. 1. Microbial Rhodopsins: Phylogenetic and Functional Diversity (J. Spudich & K. Jung). 2. Sensory Rhodopsin Signaling in Green Flagellate Algae (O. Sineshchekov & J. Spudich). 3. Visual Pigments as Photoreceptor (M. Kumauchi & T. Ebrey). 4. Structural and Functional Aspects of the Mammalian Rod-Cell Photoreceptor Rhodopsin (N. Abdulaev & K. Ridge). 5. A Novel Light-Sensing Pathway in the eye: Conserved Features of Inner Retinal Photoreception in Rodents, Man, and Teleost Fish (M. Hankins & R. Foster). 6. The Phytochromes (S. Tu & J. Lagarias). 7. Phytochrome Signaling (E. Huq & P. Quail). 8. Phytochromes in Microorganisms (R. Vierstra & B. Karniol). 9. Light-activated Intracellular Movement of Phytochrome (E. Schafer & F. Nagy). 10. Plant Cryptochromes: Their Genes, Biochemistry, and Physiological Roles (A. Batschauer). 11. Plant Cryptochromes and Signaling (A. Cashmore). 12. Animal Cryptochromes (R. van Gelder & A. Sancar). 13. Blue-Light Sensing and Signaling by the Phototropins (J. Christie & W. Briggs). 14. LOV-domain Photochemistry (T. Swartz & R. Bogomolni). 15. LOV-domain Structure, Dynamics, and Diversity (S. Crosson). 16. The ZEITLUPE Family of Putative Photoreceptors (T. Schultz). 17. Photoreceptor Gene Families in Lower Plants (N. Suetsugu & M. Wada). 18. Neurospora Photoreceptors (J. Dunlap & J. Loros). 19. Photoactive Yellow Protein, THE Xanthopsin (M. van der Horst, et al.). 20. Hypericin-Like Photoreceptors (P. Song). 21. The Antirepressor AppA Uses the Novel Flavin-Binding BLUF domain as a Blue-Light-Absorbing Photoreceptor to Control Photosystem Synthesis (S. Masuda & C. Bauer). Discovery, Characterization, and Prospect of Photoactivated Adenylyl Cyclase (PAC), a Novel Blue-Light Receptor Flavoprotein from Euglena gracilis (M. Watanabe & M. Iseki).

Demonstration of a sensory rhodopsin in eubacteria

We report the first sensory rhodopsin observed in the eubacterial domain, a green light‐activated photoreceptor in Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. The gene encoding the membrane opsin protein of 261 residues (26 kDa) and a smaller gene encoding a soluble protein of 125 residues (14 kDa) are under the same promoter in a single operon. The opsin expressed heterologously in Escherichia coli membranes bound all‐trans retinal to form a pink pigment (λmax 543 nm) with a photochemical reaction cycle of 110 ms half‐life (pH 6.8, 18°C). Co‐expression with the 14 kDa protein increased the rate of the photocycle, indicating physical interaction with the membrane‐embedded rhodopsin, which we confirmed in vitro by affinity enrichment chromatography and Biacore interaction. The pigment lacks the proton donor carboxylate residue in helix C conserved in known retinylidene proton pumps and did not exhibit detectable proton ejection activity. We detected retinal binding to the protein in Anabaena membranes by SDS‐PAGE and autofluorography of 3H‐labelled all‐trans retinal of reduced membranes from the organism. We conclude that Anabaena rhodopsin functions as a photosensory receptor in its natural environment, and suggest that the soluble 14 kDa protein transduces a signal from the receptor. Therefore, unlike the archaeal sensory rhodopsins, which transmit signals by transmembrane helix–helix interactions with membrane‐embedded transducers, the Anabaena sensory rhodopsin may signal through a soluble cytoplasmic protein, analogous to higher animal visual pigments.

New Insights into Metabolic Properties of Marine Bacteria Encoding Proteorhodopsins

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.

Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics

Silencing neurons using optogenetics Rhodopsin light-sensitive ion channels from green algae provide a powerful tool to control neuronal circuits. Rhodopsin cation channels effectively depolarize neurons and cause the firing of short-lived electrical membrane potentials. Govorunova et al. describe algal channels that do the opposite; that is, they hyperpolarize or silence particular neurons (see the Perspective by Berndt and Deisseroth). These cation channels provide greater light sensitivity than that of existing hyperpolarizing light-activated channels, operate rapidly, and selectively conduct only anions. This approach is an ideal complement to the widely used technique of creating light-sensitive neurons through the expression of rhodopsin cation channels. Science, this issue p. 647; see also p. 590 An anion channel from algae allows optogenetic silencing of neurons. [Also see Perspective by Berndt and Deisseroth] Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins

The archaeal rhodopsins are a family of seven‐transmembrane‐helix, visual pigment‐like proteins found in Halobacterium salinarum and related halophilic Archaea. Two, bacteriorhodopsin (BR) and halorhodopsin (HR), are transport rhodopsins that carry out light‐driven electrogenic translocation of protons and chloride, respectively, across the cell membrane. The other two, sensory rhodopsins I and II (SRI and SRII), are phototaxis receptors that send signals to tightly bound transducer proteins that in turn control a phosphorylation cascade modulating the cells flagellar motors. Recent progress has cast light on how nature has modified the common design of these proteins to carry out their distinctly different functions: electrogenic ion transport and non‐electrogenic signal transduction. A key shared mechanism between BR and SRII appears to be an interhelical salt bridge locked conformational switch that is released by photoisomerization of retinal. In BR disruption of the lock opens a cytoplasmic half‐channel that ensures uptake of the transported proton from the cytoplasmic side of the membrane at a critical time in the pumping cycle. Transducer‐free SRI uses the same mechanism to carry out light‐driven proton transport, but interaction with its transducer blocks the cytoplasmic half‐channel thereby interrupting the transport cycle. In SRI, transducer interaction also disrupts the salt bridge in the dark, poising the receptor in an intermediate conformation able to produce opposite signals depending on the colour of the stimulus light. A model for signalling is proposed in which the salt bridge‐controlled half‐channel is used to modulate interaction with the Htr proteins when the receptor signalling states are formed.

Spectroscopic and Photochemical Characterization of a Deep Ocean Proteorhodopsin

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20–30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltagegated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family.

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 retinals photoisomerized C13C14 double bond to residues in the membrane-embedded α-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRIIs 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.