Plant and Cell Physiology Advance Access originally published online on September 4, 2008
Plant and Cell Physiology 2008 49(10):1600-1606; doi:10.1093/pcp/pcn132
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Crucial Role in Light Signal Transduction for the Conserved Met93 of the BLUF Protein PixD/Slr1694
1 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501 Japan
2 Advancesoft Corporation, Akasaka, Tokyo. 107-0052 Japan
3 Center for Biological Resources and Informatics, Research Center for the Evolving Earth and Planets, Tokyo Institute of Technology, Yokohama, 226-8501 Japan
4 Department of Biomolecular Functional Engineering, Ibaraki University, Hitachi, 316-8511 Japan
*Corresponding author: E-mail, shmasuda{at}bio.titech.ac.jp; Fax, +81-45-924-5823.
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PixD/Slr1694 from the cyanobacterium Synechocystis sp. PCC6803 is a member of a new class of flavin-containing blue-light sensory proteins containing a BLUF (blue light using flavin) domain. The photocycle reaction mechanism of BLUF is unique because only small structural changes of a bound chromophore are accompanied by a few hydrogen bond rearrangements in the chromophore-binding site. Here, we show that in PixD, Met93, the residue conserved in all BLUF domains, is crucial for light-dependent signal transduction. Specifically, the light-insensitive M93A mutant of PixD revealed biochemical and physiological activities compatible with those of the light-adapted wild-type PixD. However, the W91A mutant of PixD retained light sensitivity and biological function, although the corresponding mutant of another BLUF protein, AppA, has been reported to be locked in the light signaling state. These observations suggest that the pathway through which the light signal is transformed into apoprotein structural changes has been modified in BLUF proteins for their respective functions.
Keywords: BLUF - Cyanobacteria - Flavin - Photoreceptor - PixD
Abbreviations: BLUF, blue-light using flavin; FTIR, Fourier-transform infrared.
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Introduction |
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Photoreceptor proteins are important in most organisms for adaptation of their physiology to environmental light conditions. These photoreceptors sense specific wavelengths of light, and transmit the light signal into downstream components. Although mechanisms of light signal transduction initiated by light excitation of the bound chromophore (the photocycle reaction) have been proposed for most photoreceptors, the molecular details for translation of the light signal into a change in protein structure are still largely unknown.
Sensors of blue light using flavin (BLUF) proteins are a new member of the photoreceptor family that is widely conserved in eukaryotic and prokaryotic microorganisms (Gomelsky and Klug 2002
, van der Horst and Hellingwerf 2004, Masuda and Bauer 2005, Kennis and Groot 2007, Losi 2007). BLUF proteins that have been characterized include AppA in the purple bacterium Rhodobacter sphaeroides (Masuda and Bauer 2002), PAC in the alga Euglena gracilis (Iseki et al. 2002) and PixD (also called Slr1694 or Tll0078) in the cyanobacteria Synechocystis sp. PCC6803 and Thermosynechococcus elongatus (Masuda et al. 2004, Okajima et al. 2005). AppA works as a light-dependent anti-repressor for transcription of photosynthesis genes (Gomelsky and Kaplan 1995, Braatsch et al. 2002, Masuda and Bauer 2002). PAC is a blue-light activated adenylyl cyclase that is involved in the photoavoidance response (Iseki et al. 2002). PixD is involved in photophobic regulation of pili-dependent cell motility (Masuda and Ono 2004, Okajima et al. 2005). According to yeast two-hybrid analysis (Okajima et al. 2005), Synechocystis PixD interacts with a PatA-like response regulator of the bacterial two-component system PixE, which may control activity of pili to modulate phototactic behaviors.
The photocycle reaction mechanism of BLUF has been extensively studied with AppA. These studies have suggested that a proton-coupled electron transfer occurs from Tyr21 (Fig. 1B) to the bound flavin upon light excitation of flavin, which results in the formation of a flavin radical that causes the formation of a new hydrogen bond with flavin O4 presumably due to rotation of the Gln63 side chain by 
180° (Anderson et al. 2005, Dragnea et al. 2005, Gauden et al. 2005, Masuda et al. 2005b; Grinstead et al. 2006a, Unno et al. 2006, Gauden et al. 2007, Domratcheva et al. 2008). The light-induced proton-coupled electron transfer was also established in PixD (Gauden et al. 2006), although some other amino acids may be involved in the electron transfer (Fukushima et al. 2008). The additional hydrogen bond to flavin O4 leads to a red shift in flavin absorption as well as alteration of the Trp104 position, which causes specific changes in β-sheet structure to transmit the signal towards a downstream component, the transcriptional repressor PpsR (Masuda et al. 2005c, Grinstead et al. 2006b). This model has recently been confirmed by in vivo and in vitro site-directed mutagenesis analyses of AppA (Masuda et al. 2007). However, because the tryptophan residue (Trp104 of AppA) is not completely conserved in all BLUF domains identified (e.g. YcgF of Escherichia coli) (Gomelsky and Klug 2002), it is still unknown whether the proposed model for the AppA-like intramolecular signaling path is common among other BLUF proteins, including PixD. In fact, several other alternative models have been proposed recently for the mechanisms of the light signal transduction of BLUF proteins (discussed later).
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Recently, crystal structures of PixD from T. elongatus BP-1 (Tll0078) and Synechocystis sp. PCC6803 (Slr1694) have been determined (Kita et al. 2005
, Yuan et al. 2006). Both crystal forms contain a decamer in the asymmetric unit with two pentameric rings stacked face-to-face. The overall structures of the BLUF core domain of PixDs are very similar to each other and also to that of AppA (Anderson et al. 2005, Jung et al. 2006). In the crystal structures of PixDs, Trp91 (which corresponds to Trp104 of AppA) has swung away from the flavin moiety, and Met93 is positioned near the flavin to form a hydrogen bond with the amide side chain of Gln50 (Kita et al. 2005, Yuan et al. 2006) (which corresponds to Gln63 of AppA) (Fig. 1) as reported in BlrB (Jung et al. 2005) and C20S mutant AppA (Jung et al. 2006), with the exception for one of 10 crystallographic subunits of Synechocystis PixD, in which Trp91 and Met93 are positioned near the flavin arrangement, similar to that of wild-type AppA (Fig. 1B). The different localization of Trp91 and Met93 in crystals of PixDs as well as among BLUF proteins suggests that the dynamic properties of these two amino acids play important roles during light signal transduction by the protein. However, as yet few studies have been conducted to elucidate the functional role of these amino acids. In this study, to clarify the functional role of these amino acids, we characterized the properties of site-directed mutants of Synechocystis PixD both in vitro and in vivo. |
Results and Discussion |
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We previously applied light-induced Fourier transform infrared (FTIR) difference spectroscopy to characterize light-induced structural changes of PixD (Hasegawa et al. 2004
, Masuda et al. 2004, Hasegawa et al. 2005). These studies identified several distinct structural changes in both chromophore and apoprotein upon light excitation. In this study, to assess the effects of the mutations on the light-dependent structural changes of PixD, we first measured light-minus-dark difference FTIR spectra of W91A and M93A mutant proteins. As shown in Fig. 2A, the spectral features of W91A mutant protein (red line) were largely similar to those of the wild-type spectrum (black line), indicating that light-dependent structural changes of the chromophore and apoprotein are not modified much in the W91A mutant. Based on our previous analysis, the 1713(–)/1697(+) cm–1 bands in the mutant spectrum can be ascribed to the C4=O stretch vibration of the flavin isoalloxazine ring (Masuda et al. 2004, Hasegawa et al. 2005), and the downshift of the band from 1713 to 1697 cm–1 upon light excitation indicates the strengthened hydrogen bonding to flavin O4 upon light irradiation, although the light state band of the mutant (1697 cm–1) was upshifted by 4 cm–1 compared with the wild type (1693 cm–1). In contrast, the FTIR spectrum was significantly altered upon M93A mutation (Fig. 2B). Despite the normal appearance of the C4=O bands, the prominent bands around 1550(+) to 1510(–) cm–1 observed in the wild-type spectrum were not detected in the M93A mutant spectrum. These bands have been primarily attributed to amide II modes, due to the NH deformations and CN stretches of polypeptide backbones (Masuda et al. 2004, Hasegawa et al. 2005, Hasegawa et al. 2006). These results indicate that Met93, but not Trp91, is necessary for inducing the light-dependent structural changes of the apoprotein inferred from the FTIR.
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To gain more insight into the relationship between light-induced structural changes and function in PixD, we characterized wild-type and mutant PixDs biochemically. First, we analyzed the interaction of PixDs and PixE using a pull-down assay with the purified proteins. For this analysis, His-tagged PixE was bound to an Ni column, and then untagged PixD was applied to the column (Fig. 3, Input). The column was washed once with buffer, and then the proteins bound to the Ni column were eluted with buffer containing 1 M imidazole (Fig. 3, Output). Under dark conditions (D), wild-type PixD was specifically associated with PixE, and the association was completely abolished by light irradiation (L), showing that PixD directly interacts with PixE in a light-dependent manner. In contrast, Y8F and M93A mutant PixD did not interact with PixE under either dark or light-illuminated conditions, suggesting that both mutant proteins are functionally locked in the light signaling state. The Y8F mutation results in total loss of light-induced spectral changes in both visible light and FTIR spectra (Hasegawa et al. 2005
), indicating that the mutant protein does not respond to blue light as reported in other BLUF proteins (Kraft et al. 2003, Fukushima et al. 2008). Notably, the M93A mutant protein still shows a light-induced flavin absorption red shift (Yuan et al. 2006) and changes in the C4=O stretch vibration (Fig. 2B), indicating that the light-induced changes in the flavin chromophore are decoupled from signaling state formation. However, W91A mutant PixD is associated with PixE under dark conditions, and light irradiation markedly weakened the interaction between W91A mutant PixD and PixE as observed for wild-type protein. The result suggests that W91A mutant PixD responds to blue light, although the interaction was not completely abolished. The residual interaction can be accounted for by the partial existence of the dark state protein during illumination at the intensity used due to the markedly enhanced dark decay of the signaling state (5.7-fold as compared with that of the wild type) observed in the mutant protein (Yuan et al. 2006). These results indicate that the effects of M93A mutation are not achieved through Trp91 (corresponding to Trp104 in AppA). Notably, the M93A mutation, but not the W91A mutation, resulted in loss of the amide bands including the 1,544(+)/1,510(–) cm–1 bands that are attributable to apoprotein structural changes (Fig. 2), suggesting that these structural changes are directly responsible for the light-dependent PixD–PixE interaction to form the signaling state.
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Spectroscopic and biochemical analyses of the mutant PixDs led to the prediction that Met93 has prominent roles in the in vivo mechanism of light-dependent signal transduction by PixD. Next we further analyzed the effects of the mutations on the physiological behavior of Synechocystis cells. For this analysis, DNA fragments coding for wild-type and for Y8F, W91A and M93A mutant PixD fused with promoter DNA were cloned into an IncQ plasmid, pJRD215 (see Materials and Methods). Then, these plasmid constructs were separately introduced into a PixD null mutant, and the phenotype of each complementing strain was examined. Given that the copy number of IncQ plasmids in Synechocystis cells could not be much higher than that of the the chromosome (
12 copies per cell) (Kreps et al. 1990), in vivo protein levels of PixD in the complementing strains must be similar to those in the wild type. The PixD null mutant shows phototactic motility away from a light source (negative phototaxis), while the wild-type strain shows positive phototaxis (Masuda and Ono 2004, Okajima et al. 2005). As shown in Fig. 4, the complementation strain with the W91A mutant pixD showed positive phototaxis as observed in the wild-type strain. However, complementation strains with Y8F and M93A mutant pixD showed negative phototaxis as observed in the PixD null mutant, although the copy number of pixD, encoded on the plasmid, in each complementing strain may differ slightly from that in the wild-type strain. These results indicate that the W91A mutant, but not the Y8F or M93A mutant, can compensate for loss of function of PixD in vivo. It is of note in this context that blue light did not induce any phototaxis response of this bacterium, presumably due to mutual effects of photoreceptors other than PixD through a complex signaling network (Okajima et al. 2005). Thus, the phototactic assay was performed by using monochromatic light at 660 nm, a wavelength at which the flavin chromophore is not excited, for simplicity, so that PixD must be preserved in its dark state during the assay. As wild-type and W91A mutant PixDs interact with PixE, and as the Y8F and M93A mutant PixD do not associate with PixE in the dark (Fig. 3), it is most likely that the positive phototaxis is ascribed to PixE that is bound to PixD, and that Y8F and M93A mutants are stationary in the state corresponding to the light signaling state for wild-type PixD.
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The results obtained in this study clearly indicate that Met93 but not Trp91 is crucial for the physiological function of Synechocystis PixD. We previously showed by in vivo and in vitro site-directed mutagenesis analysis that Trp104 (Trp91 of PixD) is crucial for the biochemical function of AppA, and W104A mutant AppA is locked in the light signaling state even in the dark (Masuda et al. 2007
). Therefore, these and the present results may be interpreted such that the intramolecular pathway for signal transduction has apparently been specifically modified between these two BLUF photoreceptors; a pathway via Met93 in PixD and via Trp104 in AppA, respectively. However, at present, we cannot completely exclude the possibility that both pathways are functional to different extents in AppA and PixD, and that an equivalent point mutation results in different effects on the pathways depending on the proteins. The highly conserved tryptophan (Trp104 of AppA) or methionine (Met93 of PixD) exist on the β5 strand that acts as a transducer in light signal transduction of BLUF proteins (Masuda et al. 2005c, Grinstead et al. 2006b, Jung et al. 2006). Thus, the signal output mediated by the different residue may be specifically modified, although the formation of a flavin radical is the common initial photochemical step conserved in all BLUF proteins. The proposed difference in the intramolecular signaling pathway may provide competence of each BLUF protein for wide varieties of physiological functions.
Several spectroscopic analyses have established that hydrogen bonding toward flavin O4 is strengthened in the signaling state of the BLUF proteins (Kraft et al. 2003, Laan et al. 2003, Hasegawa et al. 2004, Masuda et al. 2004, Gauden et al. 2005, Hasegawa et al. 2005, Masuda et al. 2005a, Masuda et al. 2005b, Unno et al. 2005, Zirak et al. 2005, Grinstead et al. 2006a, Grinstead et al. 2006b, Hasegawa et al. 2006, Unno et al. 2006, Majerus et al. 2007, Stelling et al. 2007, Takahashi et al. 2007). However, the detailed molecular mechanism for the signaling state formation is still a matter of debate, and four different models have now been proposed by several groups. The first one is that the amide side chain of Gln50 (Gln63 of AppA) rotates by 180° upon light illumination to form a new hydrogen bond with flavin O4 in the signaling state, in which the structures of Fig. 1A and B represent the light and dark states, respectively (Anderson et al. 2005, Gauden et al. 2006, Grinstead et al. 2006a, Unno et al. 2006, Yuan et al. 2006, Masuda et al. 2007). The second one is that the side chain of Gln50 (Gln63 of AppA) is tautomerized and rotates by 180° to form a strong hydrogen bond with flavin O4 in the signaling state, in which the structures of Fig. 1A and B represent the dark and light states, respectively (Domratcheva et al. 2008). The third model is that the conserved glutamine is tautomerized upon light illumination to form a new hydrogen bond with flavin O4 without the rotation of the glutamine side chain, and its amide side chain makes a hydrogen bond with the conserved tyrosine (Tyr21 of AppA) in both the dark and light states, as shown in Fig. 1B (Stelling et al. 2007). The fourth model is that the conserved glutamine does not rotate upon light illumination and the glutamine amide side chain as well as tryptophan (Trp104 of AppA) make hydrogen bonds with flavin O4 in the signaling state (Obanayama et al. 2008). Instead, the distance and the bond angle between the glutamine side chain amide and flavin O4 are suggested to alter upon light illumination to strengthen the hydrogen bond toward flavin O4, in which Fig. 1A represents the dark state structure but Fig. 1B is not the exact light state structure, although they share some characteristics (Takahashi et al. 2007, Obanayama et al. 2008). At present, available evidence seems to be insufficient to establish which model reflects the correct mechanism.
The present results indicate that M93A mutation locked PixD in the light signaling state as found in the W104A mutant of AppA. This seems to support the idea that there is a hydrogen bond between Gln50 and Met93 in dark-adapted PixD (as in Fig. 1A), and the loss of the hydrogen bond upon M93A mutation results in a conformational change compatible with the light signaling state. Furthermore, this view is not in contradiction to the observation that the postulated dark state structure was found in nine of the 10 subunits of Synechocystis PixD crystals (Yuan et al. 2006) and in all subunits of T. elongatus PixD crystals (Kita et al. 2005), which were formed in the dark. The Y8F mutant protein was also locked in the light signaling state (Figs. 3, 4), suggesting that the arrangement of the hydrogen bond network including Try8, Gln50 and Met93 is crucial for arrest of the BLUF domain in the dark state structure.
Although the PixD structure shown in Fig. 1A is likely to reflect the hydrogen bond network for dark state PixD, there is some ambiguity as to whether the lower panel (Fig. 1B) structure reflects the structure of the light signaling state of PixD. FTIR analyses showed that the spectral features of the light-induced changes in C4=O stretch vibration are not influenced by the M93A mutation (Fig. 2B), indicating that Met93 may not be involved in this change directly. In contrast, the light state C4=O band, but not the dark state band, was upshifted upon W91A mutation (Fig. 2A). This may suggest that Trp91 participates in hydrogen bonding towards flavin O4 in the light signaling state, but this hydrogen bond is not directly involved in the light-induced spectral changes for C4=O. This view is not in contradiction to results from recent tryptophan fluorescence experiments that suggested that the conserved tryptophan (Trp91 of PixD) is in a buried conformation even in the signaling state (Toh et al. 2008). As the Gln50 side chain amide seems to form hydrogen bonds with flavin N5 and O4 as well as Met93 under dark conditions (Fig. 1A), it may be possible that, in the light signaling state of PixD, the Gln50 side chain is tautomerized (as in the second model) (Domratcheva et al. 2008) or the distance between Gln50 and flavin O4 may be shortened by unknown mechanisms (as in the fourth model) (Takahashi et al. 2007, Obanayama et al. 2008), which results in strengthened hydrogen bonding to O4. This reaction may lead to switching of the position of Met93, which is the structural determinant controlling the interaction of PixD and PixE.
Notably, 1544(+)/1510(–) cm–1 amide bands in the FTIR spectra could be attributable to structural changes of the apoprotein in the signaling state, because these bands were absent in the M93A mutant spectrum (Fig. 2B). Further detailed spectroscopic, structural and biochemical analyses coupled with physiological experiments with other BLUF proteins will identify the exact features of the signal relay, which will also give us an overview of the structural basis of the molecular evolution of biological photoreceptors.
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Protein purification
The pixE (slr1693) cording region of Synechocystis sp. PCC6803 was amplified by PCR using isolated genomic DNA as a template, and forward (5'- GGGCCCGCATATGAGCAATTCAGTTTTGTCC-3') and reverse (5'-GGGGGGGGTCGACGGAGTTGGTTTTATTGGTG-3') primers. The amplified fragment was cloned into the NdeI and SalI sites of the pET29(a) vector (Merck, Nottingham, UK). The resulting plasmid, named pETSlr1693, was transferred into an E. coli strain BL21(DE3) (Merck). The His-tagged PixE protein was overexpressed by induction for >16 h at 16°C with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; TAKARA SHUZO CO., LTD., Kyoto, Japan). The expressed protein was purified with His-Bind resin (Merck) according to the manufacturer's instructions. The expression of wild-type (Masuda et al. 2004
), and Y8F (Hasegawa et al. 2005), W91A and M93A mutant (Yuan et al. 2006) PixD proteins was carried out as described.
In vitro pull-down assay
All experiments were performed at room temperature. The purified His-tagged PixE (50 µg) was incubated with 0.5 ml (bed volume) of His-Bind resin (Merck) for 10 min in a buffer contaning 10 mM Tris–HCl (pH 7.5) and 100 mM NaCl. The resin was then set on the Poly-Prep Column (Bio-Rad, Hercules, CA, USA). The purified wild-type, Y8F, W91A or M93A mutant PixD (with the concentrations adjusted to 45 µM with the same buffer) was applied to the column five times under the dark or light-illuminated (1,500 µmol photons m–2 s–1) conditions. After washing the resin with 0.5 ml of the same buffer, proteins were eluted with the buffer containing 1 M imidazole. The eluted proteins were analyzed by SDS–PAGE.
Complementation analysis
The pixD promoter region was amplified by PCR using isolated genomic DNA as a template, and forward (5'-GTTGAATTCTCCCCCCAATC-3') and reverse (5'-GGGGGGCATATGTGTGCGTAGCTTTTAGCT-3') primers. The amplified fragment was digested with EcoRI and NdeI, and then cloned into an EcoRI site of the pJRD215 plasmid (Davison et al. 1987) with an EcoRI–NdeI-digested insert DNA (PixD coding region) of pTYSlr1694 (wild type) (Masuda et al. 2004), pTY8F (W8F mutant) (Hasegawa et al. 2005), pTYSlr1694-W91A (W91A mutant) or pTYSlr1694-M93A (M93A mutant) (Yuan et al. 2006). The resulting plasmids, designated as pJRD-WT, pJRD-Y8F, pJRD-W91A and pJDR-M93A, respectively, were transferred into the pixD null mutant (
PixD) (Masuda and Ono 2004) by conjugation with an E. coli strain S17-1 (Simon et al. 1983). The phototactic assay was carried out essentially as described (Masuda and Ono 2004). Briefly, cell cultures of the complementing strains were spotted separately onto agar-solidified plates (the positions of spots are highlighted by white circles in Fig. 4), and cells were incubated under lateral illumination with a red light (660 nm) provided by a light-emitting diode (Sanyo, Tokyo, Japan).
FTIR spectroscopy
Sample preparations and FTIR measurements were carried out essentially as described (Masuda et al. 2004, Hasegawa et al. 2005).
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The Research Foundation for Opto-Science & Technology (to S.M.); the Ministry of Education, Culture, Science and Technology of Japan (to S.M., H.O. and T.-A.O).
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References |
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Anderson S, Dragnea V, Masuda S, Ybe J, Moffat K, Bauer C. Structure of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides. Biochemistry (2005) 44:7998–8005.[CrossRef][Web of Science][Medline]
Braatsch S, Gomelsky M, Kuphal S, Klug G. A single flavoprotein, AppA, integrates both redox and light signals in Rhodobacter sphaeroides. Mol. Microbiol (2002) 45:827–836.[CrossRef][Web of Science][Medline]
Davison J, Heusterspreute M, Chevalier N, Ha-Thi V, Brunel F. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene (1987) 51:275–280.[CrossRef][Web of Science][Medline]
Domratcheva T, Grigorenko BL, Schlichting I, Nemukhin AV. Molecular models predict light-induced glutamine tautomerization in BLUF photoreceptors. Biophys. J (2008) 94:3872–3879.[CrossRef][Web of Science][Medline]
Dragnea V, Waegele M, Balascuta S, Bauer C, Dragnea B. Time-resolved spectroscopic studies of the AppA blue-light receptor BLUF domain from Rhodobacter sphaeroides. Biochemistry (2005) 44:15978–15985.[CrossRef][Web of Science][Medline]
Fukushima Y, Murai Y, Okajima K, Ikeuchi M, Itoh S. Photoreactions of Tyr8- and Gln50-mutated BLUF domains of the PixD protein of Thermosynechococcus elongatus BP-1: photoconversion at low temperature without Tyr8. Biochemistry (2008) 47:660–669.[CrossRef][Web of Science][Medline]
Gauden M, Grinstead JS, Laan W, van Stokkum IH, Avila-Perez M, Toh KC, Boelens R, Kaptein R, van Grondelle R, Hellingwerf KJ, Kennis JT. On the role of aromatic side chains in the photoactivation of BLUF domains. Biochemistry (2007) 46:7405–7415.[CrossRef][Web of Science][Medline]
Gauden M, van Stokkum IH, Key JM, Luhrs D, van Grondelle R, Hegemann P, Kennis JT. Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor. Proc. Natl Acad. Sci. USA (2006) 103:10895–10900.
Gauden M, Yeremenko S, Laan W, van Stokkum IH, Ihalainen JA, van Grondelle R, Hellingwerf KJ, Kennis JT. Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes. Biochemistry (2005) 44:3653–3662.[CrossRef][Web of Science][Medline]
Gomelsky M, Kaplan S. appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J. Bacteriol (1995) 177:4609–4618.
Gomelsky M, Klug G. BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms. Trends Biochem. Sci (2002) 27:497–500.[CrossRef][Web of Science][Medline]
Grinstead JS, Avila-Perez M, Hellingwerf KJ, Boelens R, Kaptein R. Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain. J. Am. Chem. Soc (2006a) 128:15066–15067.[CrossRef][Web of Science][Medline]
Grinstead JS, Hsu ST, Laan W, Bonvin AM, Hellingwerf KJ, Boelens R, Kaptein R. The solution structure of the AppA BLUF domain: insight into the mechanism of light-induced signaling. Chembiochem (2006b) 7:187–193.[CrossRef][Web of Science][Medline]
Hasegawa K, Masuda S, Ono TA. Structural intermediate in the photocycle of a BLUF (sensor of blue light using FAD) protein Slr1694 in a Cyanobacterium Synechocystis sp. PCC6803. Biochemistry (2004) 43:14979–14986.[CrossRef][Web of Science][Medline]
Hasegawa K, Masuda S, Ono TA. Spectroscopic analysis of the dark relaxation process of a photocycle in a sensor of blue light using FAD (BLUF) protein Slr1694 of the cyanobacterium Synechocystis sp. PCC6803. Plant Cell Physiol (2005) 46:136–146.
Hasegawa K, Masuda S, Ono TA. Light induced structural changes of a full-length protein and its BLUF domain in YcgF(Blrp), a blue-light sensing protein that uses FAD (BLUF). Biochemistry (2006) 45:3785–3793.[CrossRef][Web of Science][Medline]
Iseki M, Matsunaga S, Murakami A, Ohno K, Shiga K, Yoshida K, Sugai M, Takahashi T, Hori T, Watanabe M. A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis. Nature (2002) 415:1047–1051.[CrossRef][Web of Science][Medline]
Jung A, Domratcheva T, Tarutina M, Wu Q, Ko WH, Shoeman RL, Gomelsky M, Gardner KH, Schlichting I. Structure of a bacterial BLUF photoreceptor: insights into blue light-mediated signal transduction. Proc. Natl Acad. Sci. USA (2005) 102:12350–12355.
Jung A, Reinstein J, Domratcheva T, Shoeman RL, Schlichting I. Crystal structures of the AppA BLUF domain photoreceptor provide insights into blue light-mediated signal transduction. J. Mol. Biol (2006) 362:717–732.[CrossRef][Web of Science][Medline]
Kennis JT, Groot ML. Ultrafast spectroscopy of biological photoreceptors. Curr. Opin. Struct. Biol (2007) 17:623–630.[CrossRef][Web of Science][Medline]
Kita A, Okajima K, Morimoto Y, Ikeuchi M, Miki K. Structure of a cyanobacterial BLUF protein, Tll0078, containing a novel FAD-binding blue light sensor domain. J. Mol. Biol (2005) 349:1–9.[CrossRef][Web of Science][Medline]
Kraft BJ, Masuda S, Kikuchi J, Dragnea V, Tollin G, Zaleski JM, Bauer CE. Spectroscopic and mutational analysis of the blue-light photoreceptor AppA: a novel photocycle involving flavin stacking with an aromatic amino acid. Biochemistry (2003) 42:6726–6734.[CrossRef][Web of Science][Medline]
Kreps S, Ferino F, Mosrin C, Gerits J, Mergeay M, Thuriaux P. Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803. Mol. Gen. Genet (1990) 221:129–133.[CrossRef][Web of Science]
Laan W, van der Horst MA, van Stokkum IH, Hellingwerf KJ. Initial characterization of the primary photochemistry of AppA, a blue-light-using flavin adenine dinucleotide-domain containing transcriptional antirepressor protein from Rhodobacter sphaeroides: a key role for reversible intramolecular proton transfer from the flavin adenine dinucleotide chromophore to a conserved tyrosine? Photochem. Photobiol (2003) 78:290–297.[CrossRef][Web of Science][Medline]
Losi A. Flavin-based blue-light photosensors: a photobiophysics update. Photochem. Photobiol (2007) 83:1283–1300.[CrossRef][Web of Science][Medline]
Majerus T, Kottke T, Laan W, Hellingwerf K, Heberle J. Time-resolved FT-IR spectroscopy traces signal relay within the blue-light receptor AppA. Chemphyschem (2007) 8:1787–1789.[CrossRef][Medline]
Masuda S, Bauer CE. AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell (2002) 110:613–623.[CrossRef][Web of Science][Medline]
Masuda S, Bauer CE. The antirepressor AppA uses the novel flavin-binding BLUF domain as blue-light-absorbing photoreceptor to control photosystem synthesis. In: In Handbook of Photosensory Receptors.—Briggs WR, Spudich J, eds. (2005) Wiley-VCH Verlag GmbH, Weinheim. 433–445.
Masuda S, Hasegawa K, Ishii A, Ono TA. Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of Synechocystis sp. PCC6803. Biochemistry (2004) 43:5304–5313.[CrossRef][Web of Science][Medline]
Masuda S, Hasegawa K, Ono TA. Adenosine diphosphate moiety does not participate in structural changes for the signaling state in the sensor of blue-light using FAD domain of AppA. FEBS Lett (2005a) 579:4329–4332.[CrossRef][Web of Science][Medline]
Masuda S, Hasegawa K, Ono TA. Light-induced structural changes of apoprotein and chromophore in the sensor of blue light using FAD (BLUF) domain of AppA for a signaling state. Biochemistry (2005b) 44:1215–1224.[CrossRef][Web of Science][Medline]
Masuda S, Hasegawa K, Ono TA. Tryptophan at position 104 is involved in transforming light signal into changes of β-sheet structure for the signaling state in the BLUF domain of AppA. Plant Cell Physiol (2005c) 46:1894–1901.
Masuda S, Ono TA. Biochemical characterization of the major adenylyl cyclase, Cya1, in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett (2004) 577:255–258.[CrossRef][Web of Science][Medline]
Masuda S, Tomida Y, Ohta H, Takamiya K. The critical role of a hydrogen bond between Gln63 and Trp104 in the blue-light sensing BLUF domain that controls AppA activity. J. Mol. Biol (2007) 368:1223–1230.[CrossRef][Web of Science][Medline]
Obanayama K, Kobayashi H, Fukushima K, Sakurai M. Structures of the chromophore binding sites in BLUF domains as studied by molecular dynamics and quantum chemical calculations. Photochem. Photobiol (2008) 84:1003–1010.[CrossRef][Web of Science][Medline]
Okajima K, Yoshihara S, Fukushima Y, Geng X, Katayama M, et al. Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria. J. Biochem (2005) 137:741–750.
Simon R, Priefer U, Puhler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology (1983) 1:37–45.
Stelling AL, Ronayne KL, Nappa J, Tonge PJ, Meech SR. Ultrafast structural dynamics in BLUF domains: transient infrared spectroscopy of AppA and its mutants. J. Am. Chem. Soc (2007) 129:15556–15564.[CrossRef][Web of Science][Medline]
Takahashi R, Okajima K, Suzuki H, Nakamura H, Ikeuchi M, Noguchi T. FTIR study on the hydrogen bond structure of a key tyrosine residue in the flavin-binding blue light sensor TePixD from Thermosynechococcus elongatus. Biochemistry (2007) 46:6459–6467.[CrossRef][Web of Science][Medline]
Toh KC, van Stokkum IH, Hendriks JC, Alexandre MT, Arents JC, Avila-Perez M, van Grondelle R, Hellingwerf KJ, Kennis JT. On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain. Biophys. J (2008) 95:312–321.[CrossRef][Web of Science][Medline]
Unno M, Masuda S, Ono TA, Yamauchi S. Orientation of a key glutamine residue in the BLUF domain from AppA revealed by mutagenesis, spectroscopy, and quantum chemical calculations. J. Am. Chem. Soc (2006) 128:5638–5639.[CrossRef][Web of Science][Medline]
Unno M, Sano R, Masuda S, Ono TA, Yamauchi S. Light-induced structural changes in the active site of the BLUF domain in AppA by Raman spectroscopy. J. Phys. Chem. B (2005) 109:12620–12626.[Medline]
van der Horst MA, Hellingwerf KJ. Photoreceptor proteins, ‘star actors of modern times’: a review of the functional dynamics in the structure of representative members of six different photoreceptor families. Acc. Chem. Res (2004) 37:13–20.[CrossRef][Web of Science][Medline]
Yuan H, Anderson S, Masuda S, Dragnea V, Moffat K, Bauer C. Crystal structures of the Synechocystis photoreceptor Slr1694 reveal distinct structural states related to signaling. Biochemistry (2006) 45:12687–12694.[CrossRef][Web of Science][Medline]
Zirak P, Penzkofer A, Schiereis T, Hegemann P, Schlichting I. Absorption and fluorescence spectroscopic characterization of BLUF domain of AppA from Rhodobacter sphaeroides. Chem. Phys (2005) 315:142–154.[CrossRef][Web of Science]
(Received August 3, 2008; Accepted September 1, 2008)
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