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Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process., (open access.)

Small RNAs (sRNAs) form complexes with Argonaute proteins and bind to transcripts with complementary sequences to repress gene expression. sRNA-mediated regulation is conserved in a diverse range of eukaryotes and is involved in the control of various physiological functions. sRNAs are present in the unicellular green alga Chlamydomonas reinhardtii , and genetic analyses revealed that the core sRNA biogenesis and action mechanisms are conserved with those of multicellular organisms. However, the roles of sRNAs in this organism remain largely unknown. Here, we report that Chlamydomonas sRNAs contribute to the induction of photoprotection. In this alga, photoprotection is mediated by LIGHT HARVESTING COMPLEX STRESS-RELATED 3 (LHCSR3), whose expression is induced by light signals through the blue-light receptor phototropin (PHOT). We demonstrate here that sRNA-defective mutants showed increased PHOT abundance leading to greater LHCSR3 expression. Disruption of the precursor for two sRNAs predicted to bind to the PHOT transcript also increased PHOT accumulation and LHCSR3 expression. The induction of LHCSR3 in the mutants was enhanced by light containing blue wavelengths, but not by red light, indicating that the sRNAs regulate the degree of photoprotection via regulation of PHOT expression. Our results suggest that sRNAs are involved not only in the regulation of photoprotection but also in biological phenomena regulated by PHOT signaling.

Phototropin (phot) is a blue light sensor involved in the light responses of several species from green algae to higher plants. Phot consists of two photoreceptive domains (LOV1 and LOV2) and a Ser/Thr kinase domain. These domains are connected by a hinge and a linker domain. So far, studies on the photochemical reaction dynamics of phot have been limited to short fragments, and the reactions of intact phot have not been well elucidated. Here, the photoreactions of full-length phot and of several mutants from Chlamydomonas reinhardtii ( Cr ) were investigated by the transient grating and circular dichroism (CD) methods. Full-length Cr phot is in monomeric form in both dark and light states and shows conformational changes upon photoexcitation. When LOV1 is excited, the hinge helix unfolds with a time constant of 77 ms. Upon excitation of LOV2, the linker helix unfolds initially followed by a tertiary structural change of the kinase domain with a time constant of 91 ms. The quantum yield of conformational change after adduct formation of LOV2 is much smaller than that of LOV1, indicating that reactive and nonreactive forms exist. The conformational changes associated with the excitations of LOV1 and LOV2 occur independently and additively, even when they are excited simultaneously. Hence, the role of LOV1 is not to enhance the kinase activity in addition to LOV2 function; we suggest LOV1 has different functions such as regulation of intermolecular interactions.

In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors. Red light is perceived by bilin-containing phytochromes and blue light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory light, oxygen, or voltage (LOV) domains. Photoperception spans several orders of light intensity, ranging from far below the threshold for photosynthesis to values beyond the capacity of photosynthetic CO 2 assimilation. Excess light may cause oxidative damage and cell death, processes prevented by enhanced thermal dissipation via high-energy quenching (qE), a key photoprotective response. Here we show the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii. We show that PHOT controls qE by inducing the expression of the qE effector protein LHCSR3 (light-harvesting complex stress-related protein 3) in high light intensities. This control requires blue-light perception by LOV domains on PHOT, LHCSR3 induction through PHOT kinase, and light dissipation in photosystem II via LHCSR3. Mutants deficient in the PHOT gene display severely reduced fitness under excessive light conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital role in microalgal acclimation to environments of variable light intensities.

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.

Phototropins are photoreceptors regulating the blue-light response in plants and bacteria. They consist of two LOV (light oxygen voltage sensitive) domains each containing a non-covalently bound flavin-mononucleotide (FMN) chromophore, which are connected to a serine/threonine-kinase. Upon illumination, the LOV-domains undergo conformational changes, triggering a signal cascade in the organism through kinase activation. Here, we present results from molecular dynamics simulations in which we investigate the signal transduction pathway of the wildtype LOV1-domain of Chlamydomonas reinhardtii and a methyl-mercaptan (MM) adduct of its Cys57Gly-mutant at the molecular level. In particular, we analyzed the effect of covalent-bond formation between the reactive cysteine Cys57 and the FMN-reaction center, as well as the subsequent charge redistribution, on the spatio-dynamical behavior of the LOV1-domain. We compare the calculation results with experimental data and demonstrate that these adduct state characteristics have an important influence on the response of this photosensor. The light-induced changes implicate primarily an alteration of the surface charge distribution through rearrangement of the highly flexible Cα-, Dα- and Eα-helices including the Glu51-Lys91-salt bridge on the hydrophilic side of the protein domain and a β-sheet tightening process via coupling of the Aβ- and Bβ-strands. Our findings confirm the aptitude of the LOV1-domain to function as a dimerization partner, allowing the green alga to adapt its reproduction and growth speed to the environmental conditions.

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.

The blue light photoreceptor phototropin mediates crucial processes in plants leading to optimization of photosynthesis. Phototropin comprises two flavin mononucleotide-binding LOV (light-, oxygen-, or voltage-sensitive) domains. The LOV domains undergo a photocycle upon illumination, in which two intermediates have been detected by UV/Vis spectroscopy. The triplet excited state of flavin is formed and decays within a few microseconds into a photoadduct with an adjacent cysteine, which represents the signaling state of the LOV domain. For bond formation of the photoadduct, several reaction pathways have been proposed, but evidence for an intermediate at ambient conditions has not been found. Here, we performed nanosecond time-resolved UV/Vis spectroscopy on the phototropin-LOV1 domain from Chlamydomonas reinhardtii. We designed a flow cell which was used to efficiently replace the sample after each photoexcitation because the cycling time is in the order of hundreds of seconds. The comparison of difference spectra of the wild type with those of the C57S mutant that produces only the triplet excited state revealed the existence of an additional intermediate between the triplet and the adduct state. This intermediate exhibits spectral properties similar to a neutral flavin radical. This finding supports a reaction mechanism involving a neutral radical pair., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Irradiation of the LOV1 domain from the blue-light photoreceptor phototropin of the green alga Chlamydomonas reinhardtii leads to the formation of a covalent adduct of the sulfur atom of cysteine 57 to the carbon C(4a) in the chromophore FMN. This reaction is not possible in the mutant LOV1-C57G in which this cysteine is replaced by glycine. Irradiation of LOV1-C57G in the absence of oxygen but in the presence of aliphatic mercaptans or thioethers leads to the formation of a species with an absorption maximum at 615 nm, which is identified as the neutral radical FMNH . When oxygen is admitted, the reaction is completely reversible. Irradiation of LOV1-C57G in the presence of methylmercaptan CH(3)SH under oxygen-free conditions yields, in addition to FMNH , a third species with a single absorption maximum at 379 nm. This species is stable against oxygen and is also formed when the irradiation is performed in the presence of oxygen. This species is assigned to the adduct between CH(3)SH and FMN. In aqueous solution the photoreaction of CH(3)SH with FMN leads to the fully reduced hydroquinone form FMNH(2) or its anion FMNH(-). Adduct formation apparently requires the protein cage. After formation, the adduct is stable for hours inside the protein, but decomposes immediately upon denaturation. The implications of these observations for the mechanism of adduct formation in wild type LOV domains are discussed.

The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved J alpha helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-J alpha and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the J alpha linker helix was observed in LOV1-LOV2-J alpha in agreement with previous studies of LOV2-J alpha. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the J alpha linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in helicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process.