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.

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.

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 (Thio 383 ). By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio 383 , ⌽ Thio ϭ 0.49, and the enthalpy change, ⌬H Thio ϭ 135 kJ/mol, with respect to the parent state. The formation of Thio 383 is accompanied by a considerable volume contraction, ⌬V Thio ϭ Ϫ13.5 ml/mol. Similar to phot-LOVs, Thio 383 is formed from the decay of a red-shifted transient species, T 650 , within 2 s. In both YtvA and free FMN, this transient has an enthalpy content of ϳ200 kJ/mol, and its formation is accompanied by a small contraction, ⌬V T Ϸ Ϫ1.5 ml/mol, supporting the assignment of T 650 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.

Phytochrome photoreceptors mediate light responses in plants and in many microorganisms. Here we report studies using 1 H-13 C 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.chromophore-protein interaction | signal transduction | solid-state NMR | photomorphogenesis P hytochrome was first demonstrated in plants as a red-lightdependent photoreceptor regulating numerous photomorphogenic processes (1, 2). Phytochromes are, however, also now known in photosynthetic prokaryotes including cyanobacteria (3, 4), nonphotosynthetic bacteria (5), and fungi (6). Generally, the phytochrome apoprotein binds an open-chain tetrapyrrole as a chromophore (7,8) to form the red-light absorbing Pr ground state (λ max ≈ 658 nm in case of cyanobacterial phytchrome Cph1 from Synechocystis 6803). Red light absorption photoactivates the molecule to form the photoactivated far-red-light absorbing Pfr state (λ max ≈ 702 nm for Cph1) via a series of intermediates (8-10). Photoactivation is thought to be initiated by a double bond isomerization of the chromophore (10, 11). Early NMR spectroscopic studies on proteolytic phytochrome fragments (12, 13) indicated that this isomerization occurs at the C15═C16 double bond (for numbering, see Fig. 1A), a geometrical change in line with vibrational spectroscopic investigations (14-16) and results from recent 13 C solid-state NMR (17, 18) in which the most significant changes during the light-triggered conversions are confined to rings C and D. Exact geometries of the chromophore in the Pr state have been resolved as periplanar ZZZssa configurations in bacteriophytochromes from Deinococcus radiodurans (19) and Rhodopseudomonas palustris (20) as well as in the more plant-phytochrome-like Cph1 from the cyanobacterium Synechocystis 6803 (21). On the other hand, the crystal structure of the unusual bacteriophytochrome PaBphP Pseudomonas aeruginosa (22) whose ground state is Pfr shows a ZZEssa conformation, consistent with the expected primary photochemistry at the C15═C16 double bond (Fig. 1 B vs. C). Very recently, however, Ulijasz et al. presented structural simulations based on liquid NMR data of a 20-kDa GAF (cGMP phosphodiesterase/ adenylyl cyclase/FhlA) domain fragment of "SyB-Cph1" phytochrome from the thermotolerant cyanobacterium Synechococcus OSB′ (23). Surprisingly, they concluded that photoisomerization occ...