Wolfgang Gärtner

Reporter proteins for in vivo fluorescence without oxygen

Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage–sensing domains, we engineered flavin mononucleotide–based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.

Light Modulation of Cellular cAMP by a Small Bacterial Photoactivated Adenylyl Cyclase, bPAC, of the Soil Bacterium Beggiatoa

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.

First Evidence for Phototropin-Related Blue-Light Receptors in Prokaryotes

A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants. Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs. Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio383). By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio383, Phi(Thio) = 0.49, and the enthalpy change, DeltaH(Thio) = 135 kJ/mol, with respect to the parent state. The formation of Thio383 is accompanied by a considerable volume contraction, DeltaV(Thio) = -13.5 ml/mol. Similar to phot-LOVs, Thio383 is formed from the decay of a red-shifted transient species, T650, within 2 micros. In both YtvA and free FMN, this transient has an enthalpy content of approximately 200 kJ/mol, and its formation is accompanied by a small contraction, DeltaV(T) approximately -1.5 ml/mol, supporting the assignment of T650 to the FMN triplet state, as suggested by spectroscopical evidences. These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants.

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 x 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 beta-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.

Two ground state isoforms and a chromophore D-ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

Phytochrome photoreceptors mediate light responses in plants and in many microorganisms. Here we report studies using 1H–13C magic-angle spinning NMR spectroscopy of the sensor module of cyanobacterial phytochrome Cph1. Two isoforms of the red-light absorbing Pr ground state are identified. Conclusive evidence that photoisomerization occurs at the C15-methine bridge leading to a β-facial disposition of the ring D is presented. In the far-red-light absorbing Pfr state, strong hydrogen-bonding interactions of the D-ring carbonyl group to Tyr-263 and of N24 to Asp-207 hold the chromophore in a tensed conformation. Signaling is triggered when Asp-207 is released from its salt bridge to Arg-472, probably inducing conformational changes in the tongue region. A second signal route is initiated by partner swapping of the B-ring propionate between Arg-254 and Arg-222.

Old chromophores, new photoactivation paradigms, trendy applications: flavins in blue light-sensing photoreceptors.

The knowledge on the mechanisms by which blue light (BL) is sensed by diverse and numerous organisms, and of the physiological responses elicited by the BL photoreceptors, has grown remarkably during the last two decades. The basis for this “blue revival” was set by the identification and molecular characterization of long sought plant BL sensors, employing flavins as chromophores, chiefly cryptochromes and phototropins. The latter photosensors are the foundation members of the so‐called light, oxygen, voltage (LOV)‐protein family, largely spread among archaea, bacteria, fungi and plants. The accumulation of sequenced microbial genomes during the last years has added the BLUF (Blue Light sensing Using FAD) family to the BL photoreceptors and yielded the opportunity for intense “genome mining,” which has presented to us the intriguing wealth of BL sensing in prokaryotes. In this contribution we provide an update of flavin‐based BL sensors of the LOV and BLUF type, from prokaryotic microorganisms, with special emphasis to their light‐activation pathways and molecular signal‐transduction mechanisms. Rather than being a fully comprehensive review, this research collects the most recent discoveries and aims to unveil and compare signaling pathways and mechanisms of BL sensors.

Invertebrate visual pigments.

Visual pigments (VP)? constitute a subgroup of the large protein family of G-protein-coupled signal-transducing receptors that share a common structural motif of seven membrane-spanning a-helical domains.’,2 The VP consist of a protein moiety of molecular weight of around 40 kDa with a visual chromophore covalently linked to the protein through a protonated Schiff base to a lysine residue located in the seventh transmembrane (TM) helix. The visual chromophore in vertebrates is the 1 I-cis isomer of retinal or its 3,4-dehydro derivative (A,-retinal).-? In the invertebrate kingdom, 3(3-OH) or 4-hydroxy (4-OH) retinal have been identified in addition to these c h r o m ~ p h o r e s . ~ ~ ~ Hydroxylation at position 4 is a common metabolic fate of retinoids and their parent carotenoid compounds, allowing the use of 4-OH retinal by several species. The adaptation of 3-OH retinal coincides-in evolutionary terms-with the development of the holometabolic insects whose larvae live preferentially in decaying vegetation where 3-OH carotenoids are much more abundant than the unsubstituted compounds.6 The replacement of retinal by its 3-OH derivative in the larval V P of the most recently developed insects may thus be a consequence of only plants being able to synthesize carotenoids de novo, making all animals dependent on them as the source of retinal. The distribution of 3-OH retinal in insect eyes, instead of or in addition to retinal, has been determined for a broad range of species in an attempt to find a correlation between the use of a particular chromophore and the habitat of the animal^?,^-^ It was found that hydroxylated carotenoids predominate in aqueous environments, with 3-OH retinal having been identified in several aquatic beetle^^,^ and the aquatic larvae of some dragonflies and damselflies. In-

Listening to the blue: the time-resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain

YtvA is a bacterial flavo-protein related to plant phototropin. The photochemistry of YtvA and of its isolated LOV domain (YtvA-LOV) has been characterized by optical, mass spectrometric and photocalorimetric methods. The energy content (E390) of the FMN-C4a-thiol photoadduct (YtvA390 and YtvA-LOV390) and its structural volume change (deltaV390), with respect to the parent state, have been determined by means of Laser Induced Optoacoustic Spectroscopy (LIOAS). The high value of E390, 136 and 115 kJ mol(-1), respectively, ensures a large driving force for the dark recovery to the unphotolyzed state and points to a strained conformation of the protein or/and the chromophore in the photoadduct. The value of deltaV390 is significantly different for the two proteins, deltaV390 = -12.5 ml mol(-1) in YtvA and -17.2 ml mol(-1) in YtvA-LOV. The kinetics of the dark recovery reaction for YtvA-LOV is slower than for full-length YtvA, with taurec = 3900 and 2600 s at 25 degrees C, respectively, and shows a different temperature dependence. A similarly slow kinetics can be induced in YtvA by high ionic strength. Minor differences are observed in the fluorescence and photoadduct formation quantum yield. The overall stability is higher for YtvA than for YtvA-LOV. The data as a whole are indicative of an interaction between the two domains of YtvA, most probably mediated by electrostatic interactions that renders the full-length protein a compact and more rigid unit. The results reported here support the idea that the formation of the photoadduct changes the dynamics of the protein, depending on the conformational flexibility of the parent state. Flashing of the photoadduct induces a negligible deltaV, with 96% of the excitation energy dissipated as heat in <20 ns, indicating that the photoadduct does not undergo a photocycle on the LIOAS time scale, or that the photoinduced reactions occur with very low yield.

Distribution and Phylogeny of Light-Oxygen-Voltage-Blue-Light-Signaling Proteins in the Three Kingdoms of Life†

Plants and fungi respond to environmental light stimuli via the action of different photoreceptor modules. One such class, responding to the blue region of light, is constituted by photoreceptors containing so-called light-oxygen-voltage (LOV) domains as sensor modules. Four major LOV families are currently identified in eukaryotes: (i) the plant phototropins, regulating various physiological effects such as phototropism, chloroplast relocation, and stomatal opening; (ii) the aureochromes, mediating photomorphogenesis in photosynthetic stramenopile algae; (iii) the plant circadian photoreceptors of the zeitlupe (ZTL)/adagio (ADO)/flavin-binding Kelch repeat F-box protein 1 (FKF1) family; and (iv) the fungal circadian photoreceptors white-collar 1 (WC-1). Blue-light-sensitive LOV signaling modules are also widespread throughout the prokaryotic world, and physiological responses mediated by bacterial LOV photoreceptors were recently reported. Thus, the question arises as to the evolutionary relationship between the pro- and eukaryotic LOV photoreceptor systems. We used Bayesian and maximum-likelihood tree reconstruction methods to infer evolutionary scenarios that might have led to the widespread appearance of LOV domains among the pro- and eukaryotes. The phylogenetic study presented here suggests a bacterial origin for the LOV domains of the four major eukaryotic LOV photoreceptor families, whereas the LOV sensor domains were most likely recruited from the bacteria in the course of plastid and mitochondrial endosymbiosis.

Light-induced chromophore activity and signal transduction in phytochromes observed by 13C and 15N magic-angle spinning NMR

Both thermally stable states of phytochrome, Pr and Pfr, have been studied by 13C and 15N cross-polarization (CP) magic-angle spinning (MAS) NMR using cyanobacterial (Cph1) and plant (phyA) phytochrome sensory modules containing uniformly 13C- and 15N-labeled bilin chromophores. Two-dimensional homo- and heteronuclear experiments allowed most of the 13C chemical shifts to be assigned in both states. Chemical shift differences reflect changes of the electronic structure of the cofactor at the atomic level as well as its interactions with the chromophore-binding pocket. The chromophore in cyanobacterial and plant phytochromes shows very similar features in the respective Pr and Pfr states. The data are interpreted in terms of a strengthened hydrogen bond at the ring D carbonyl. The red shift in the Pfr state is explained by the increasing length of the conjugation network beyond ring C including the entire ring D. Enhanced conjugation within the π-system stabilizes the more tensed chromophore in the Pfr state. Concomitant changes at the ring C propionate carboxylate and the ring D carbonyl are explained by a loss of hydrogen bonding to Cph1-His-290 and transmittance of conformational changes to the ring C propionate via a water network. These and other conformational changes may lead to modified surface interactions, e.g., along the tongue region contacting the bilin chromophore.