Photochemical characterization of a novel fungal rhodopsin from Phaeosphaeria nodorum

Eukaryotic microbial rhodopsins are widespread bacteriorhodopsin-like proteins found in many lower eukaryotic groups including fungi. Many fungi contain multiple rhodopsins, some significantly diverged from the original bacteriorhodopsin template. Although few fungal rhodopsins have been studied biophysically, both fast-cycling light-driven proton pumps and slow-cycling photosensors have been found. The purpose of this study was to characterize photochemically a new subgroup of fungal rhodopsins, the so-called auxiliary group. The study used the two known rhodopsin genes from the fungal wheat pathogen, Phaeosphaeria nodorum. One of the genes is a member of the auxiliary group while the other is highly similar to previously characterized proton-pumping Leptosphaeria rhodopsin. Auxiliary rhodopsin genes from a range of species form a distinct group with a unique primary structure and are located in carotenoid biosynthesis gene cluster. Amino acid conservation pattern suggests that auxiliary rhodopsins retain the transmembrane core of bacteriorhodopsins, including all residues important for proton transport, but have unique polar intramembrane residues. Spectroscopic characterization of the two yeast-expressed Phaeosphaeria rhodopsins showed many similarities: absorption spectra, conformation of the retinal chromophore, fast photocycling, and carboxylic acid protonation changes. It is likely that both Phaeosphaeria rhodopsins are proton-pumping, at least in vitro. We suggest that auxiliary rhodopsins have separated from their ancestors fairly recently and have acquired the ability to interact with as yet unidentified transducers, performing a photosensory function without changing their spectral properties and basic photochemistry.     

The xanthopsins: a new family of eubacterial blue-light photoreceptors.

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.     

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.     

Photochromicity of Anabaena sensory rhodopsin, an atypical microbial receptor with a cis-retinal light-adapted form

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin characterized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.     

Photoactive yellow protein, bacteriophytochrome, and sensory rhodopsin in purple phototrophic bacteria.

The purple photosynthetic bacteria contain a large variety of sensory and regulatory proteins, and those responding to light are among the most interesting. These currently include bacteriophytochrome (Bph), sensory rhodopsin (SR), and photoactive yellow protein (PYP), which all appear to function as light sensors. We herein interpret new findings within the context of current knowledge. For greater detail, the reader is referred to comprehensive reviews on these topics. Of the three proteins, only PYP has been well-characterized in terms of structure and physical-chemical properties in the purple bacteria, although none have well-defined functions. New findings include a cluster of six genes in the Thermochromatium tepidum genome that encodes presumed sensory rhodopsin and phototaxis proteins. T. tepidum also has a gene for PYP fused to bacteriophytochrome and diguanylate cyclase domains. The genes for PYP and its biosynthetic enzymes are associated with those for gas vesicle formation in Rhodobacter species, suggesting that one function of PYP is to regulate cell buoyancy. The association of bacteriophytochrome genes with those for reaction centers and light-harvesting proteins in Rhodopseudomonas palustris suggests that the photosynthetic antenna as well as the reaction center are regulated by Bphs. Furthermore, Rc. centenum PPR is reversibly photobleached at 702 nm rather than red-shifted as in other phytochromes, suggesting that PPR senses the intensity of white light rather than light quality. PYP from Halorhodospira(aka Ectothiorhodospira)halophila is of special interest because it has become the structural prototype for the PAS domain, a motif that is found throughout the phylogenetic tree and which plays important roles in many signaling pathways. Thus, the structural and photochemical characterization of PYP, utilizing site-directed mutagenesis, provides insights into the mechanism of signal transduction.     

Conformational coupling between the cytoplasmic carboxylic acid and the retinal in a fungal light-driven proton pump

Many fungal rhodopsins, eukaryotic structural homologues of the archaeal light-driven proton pump bacteriorhodopsin, have been discovered in the course of genome sequencing projects. Recently, two fungal rhodopsins were characterized in vitro and exhibited very different photochemical behavior. Neurospora rhodopsin possesses a slow photocycle and shows no ion transport, reminiscent of sensory rhodopsins, while Leptosphaeria rhodopsin has a fast bacteriorhodopsin-like photocycle and pumps protons light-dependently. Such a dramatic difference is surprising considering the very high degree of sequence homology of the two proteins. In this paper, we investigate whether the chemical structure of a cytoplasmic carboxylic acid, the homologue of Asp-96 of bacteriorhodopsin serving as a proton donor for the retinal Schiff base, can define the photochemical properties of fungal rhodopsins. We studied mutants of Leptosphaeria rhodopsin in which this aspartic acid was replaced with Glu or Asn using spectroscopy in the infrared and visible ranges. We show that Glu at this position is inefficient as a proton donor similar to a nonprotonatable Asn. Moreover, this replacement induces long-range structural perturbations of the retinal environment, as evidenced by changes in the vibrational bands of retinal (especially, hydrogen-out-of-plane modes) and neighboring aspartic acids and water molecules. The conformational coupling of the mutation site to the retinal may be mediated by helical rearrangements as suggested by the changes in amide and proline vibrational bands. We conclude that the difference in the photochemical behavior of fungal rhodopsins from Leptosphaeria and Neurospora may be ascribed, to some extent, to the replacement of the cytoplasmic proton donor Asp with Glu.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.     

Rhodopsin-mediated photoreception in cryptophyte flagellates

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.     

Visual and archaeal rhodopsins: similarities,differences and controversy

Rhodopsins are currently known to belong to two distinct protein families. The visual rhodopsins, found in eyes throughout the animal kingdom, are photosensory pigments. Archaeal rhodopsins, found in extreme halophiles, function as light-driven proton pumps (bacteriorhodopsins), chloride ion pumps (halorhodopsins), or photosensory receptors (sensory rhodopsins). Light absorption by rhodopsins triggers their characteristic photoconversion extending into the (milli)second time range. There are three main paradigms of rhodopsins photoconversion. (1) Initiation of the trans-cis isomerization is the very primary consequence of light absorption. (2) Rhodopsins store light energy via the charge-separation mechanism (the charge of Schiff base is separated from its counterion). (3) Full trans-cis isomerization of the chromophore is a prerequisite for the full biological activity of rhodopsins. These paradigms will be questioned.     

Crystallographic Study of the LUMI Intermediate of Squid Rhodopsin

Upon absorption of light, the retinal chromophore in rhodopsin isomerizes from the 11-cis to the trans configuration, initiating a photoreaction cycle. The primary photoreaction state, bathorhodopsin (BATHO), relaxes thermally through lumirhodopsin (LUMI) into a photoactive state, metarhodopsin (META), which stimulates the conjugated G-protein. Previous crystallographic studies of squid and bovine rhodopsins have shown that the structural change in the primary photoreaction of squid rhodopsin is considerably different from that observed in bovine rhodopsin. It would be expected that there is a fundamental difference in the subsequent thermal relaxation process between vertebrate and invertebrate rhodopsins. In this work, we performed crystallographic analyses of the LUMI state of squid rhodopsin using the P62 crystal. When the crystal was illuminated at 100 K with blue light, a half fraction of the protein was converted into BATHO. This reaction state relaxed into LUMI when the illuminated crystal was warmed in the dark to 170 K. It was found that, whereas trans retinal is largely twisted in BATHO, it takes on a more planar configuration in LUMI. This relaxation of retinal is accompanied by reorientation of the Schiff base NH bond, the hydrogen-bonding partner of which is switched to Asn185 in LUMI. Unlike bovine rhodopsin, the BATHO-to-LUMI transition in squid rhodopsin was accompanied by no significant change in the position/orientation of the beta-ionone ring of retinal.     

A Photochromic Histidine Kinase Rhodopsin (HKR1) That Is Bimodally Switched by Ultraviolet and Blue Light

Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λ_max = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λ_max = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light.     

A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment

The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacterium salinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is membrane-associated and forms with all-trans retinal a visible light-absorbing pigment with a 534 nm absorption maximum and approximately 100 nm half-bandwidth typical of retinylidene protein absorption spectra. Its lambda(max) indicates a protonated Schiff base linkage of the retinal. Laser flash kinetic spectroscopy demonstrates that the retinal-reconstituted pigment undergoes a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprotonation of the chromophore in the photocycles of bacteriorhodopsin and sensory rhodopsins I and II. The slow photocycle (seconds) and long-lived intermediates (M and O) are most similar to those of the phototaxis receptor sensory rhodopsin II. The results demonstrate a photochemically reactive member of the archaeal rhodopsin family in a eukaryotic cell.     

Evidence of microbial rhodopsins in Antarctic Dry Valley edaphic systems

Microorganisms able to synthesize rhodopsins have the capacity to translocate ions through their membranes, using solar energy to generate a proton motive force. Rhodopsins are the most abundant phototrophic proteins in oceanic surface waters and are key constituents in marine bacterial ecology. However, it remains unclear how rhodopsins are used in most microorganisms. Despite their abundance in marine and fresh‐water systems, the presence of functional rhodopsin systems in edaphic habitats has never been reported. Here, we show the presence of several new putative H⁺, Na⁺ and Cl⁺ pumping rhodopsins identified by metagenomic analysis of Antarctic desert hypolithic communities. Reconstruction of two Proteobacteria genomes harboring xanthorhodopsin‐like proteins and one Bacteroidetes genome with a Na‐pumping‐like rhodopsin indicated that these bacteria were aerobic heterotrophs possessing the apparent capacity for the functional expression of rhodopsins. The existence of these protein systems in hypolithic bacteria expands the known role of rhodopsins to include terrestrial environments and suggests a possible predominant function as heterotrophic energy supply proteins, a feasible microbial adaptation to the harsh conditions prevalent in Antarctic edaphic systems.     

Substitution of Pro206 and Ser86 residues in the retinal binding pocket of Anabaena sensory rhodopsin is not sufficient for proton pumping function

Anabaena sensory rhodopsin is a seven transmembrane protein that uses all-trans/13-cis retinal as a chromophore. About 22 residues in the retinal-binding pocket of microbial rhodopsins are conserved and important to control the quality of absorbing light and the function of ion transport or sensory transduction. The absorption maximum is 550 nm in the presence of all-trans retinal at dark. Here, we mutated Pro206 to Glu or Asp, of which the residue is conserved as Asp among all other microbial rhodopsins, and the absorption maximum and pKa of the proton acceptor group were measured by absorption spectroscopy at various pHs. Anabaena rhodopsin was expressed best in Escherichia coli in the absence of extra leader sequence when exogenous all-trans retinal was added. The wild-type Anabaena rhodopsin showed small absorption maximum changes between pH 4 and 11. In addition, Pro206Asp showed 46 nm blue-shift at pH 7.0. Pro206Glu or Asp may change the contribution to the electron distribution of the retinal that is involved in the major role of color tuning for this pigment. The critical residue Ser86 (Asp 96 position in bacteriorhodopsin: proton donor) for the pumping activity was replaced with Asp, but it did not change the proton pumping activity of Anabaena rhodopsin.     

Anticipatory active-site motions and chromophore distortion prime photoreceptor PYP for light activation

Protein photoreceptors use small-molecule cofactors called chromophores to detect light. Only under the influence of the receptors' active sites do these chromophores adopt spectral and photochemical properties that suit the receptors' functional requirements. This protein-induced change in chromophore properties is called photochemical tuning and is a prime example for the general--but poorly understood--process of chemical tuning through which proteins shape the reactivity of their active-site groups. Here we report the 0.82-A resolution X-ray structure of the bacterial light receptor photoactive yellow protein (PYP). The unusually precise structure reveals deviations from expected molecular geometries and anisotropic atomic displacements in the PYP active site. Our analysis of these deviations points directly to the intramolecular forces and active-site dynamics that tune the properties of PYP's chromophore to absorb blue light, suppress fluorescence, and favor the required light-driven double-bond isomerization.     

All-trans retinal constitutes the functional chromophore in Chlamydomonas rhodopsin

Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself.

The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 × longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a β-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.

     

Ultrafast spectroscopy of biological photoreceptors

We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogen-bond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans-cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy.     

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography

DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.     

Counterion displacement in the molecular evolution of the rhodopsin family

The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. The counterion in vertebrate rhodopsins, Glu113 in the third transmembrane helix, has an additional role as an intramolecular switch to activate G protein efficiently. Here we show on the basis of mutational analyses that Glu181 in the second extracellular loop acts as the counterion in invertebrate rhodopsins. Like invertebrate rhodopsins, UV-absorbing parapinopsin has a Glu181 counterion in its G protein-activating state. Its G protein activation efficiency is similar to that of the invertebrate rhodopsins, but significantly lower than that of bovine rhodopsin, with which it shares greater sequence identity. Thus an ancestral vertebrate rhodopsin probably acquired the Glu113 counterion, followed by structural optimization for efficient G protein activation during molecular evolution.     

Mimic of photocycle by a protein folding reaction in photoactive yellow protein

The blue light receptor photoactive yellow protein (PYP) displays rhodopsin-like photochemistry based on the trans to cis photoisomerization of its p-coumaric acid chromophore. Here, we report that protein refolding from the acid-denatured state of PYP mimics the last photocycle transition in PYP. This implies a direct link between transient protein unfolding and photosensory signal transduction. We utilize this link to study general issues in protein folding. Chromophore trans to cis photoisomerization in the acid-denatured state strongly decelerates refolding, and converts the pH dependence of the barrier for refolding from linear to nonlinear. We propose transition state movement to explain this phenomenon. The cis chromophore significantly stabilizes the acid-denatured state, but acidification of PYP results in the accumulation of the acid-denatured state containing a trans chromophore. This provides a clear example of kinetic control in a protein unfolding reaction. These results demonstrate the power of PYP as a light-triggered model system to study protein folding.