Chromophore composition of a heterologously expressed BLUF-domain

Laan, Wouter; Bednarz, Teresa; Heberle, Joachim; Hellingwerf, Klaas J.

doi:10.1039/b410923f

Cited by 59 publications

(78 citation statements)

References 20 publications

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“…It has been shown for the homologous DNA photolyase from E. coli that riboflavin and FMN fail to bind to the apoprotein (44). These findings are in contrast to other blue light receptor domains such as the light-, oxygen-, and voltagesensitive (LOV) domains of phototropin, where reconstitution with flavin analogs has been achieved (45), or the sensors of blue light using FAD (BLUF) domain, where expression conditions determine the chromophore composition (34).…”

Section: Discussioncontrasting

confidence: 48%

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Immeln

Schlesinger

Heberle

et al. 2007

Journal of Biological Chemistry

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125

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Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the lightsensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.Blue light governs many responses of organisms to environmental conditions. Cryptochromes have been shown to act as sensory blue light photoreceptors in plants and animals, with their action being as diverse as their origin (1). From sequence analysis, cryptochromes have been divided into three subgroups: animal, plant, and DASH cryptochromes (2). Animal cryptochrome synchronizes the circadian clock of Drosophila to the 24-h rhythm (3). In mammals, cryptochrome has been suggested to be involved in circadian entrainment (4), and it functions independent of light as a main component of the biological clock. Arabidopsis cryptochromes 1 and 2 (AtCRY1 2 and AtCRY2) mediate de-etiolation responses, entrain the circadian clock, trigger programmed cell death induced by singlet oxygen, and regulate flowering time, stomatal opening, and production of anthocyanin (5-8). DASH cryptochromes are mostly found in bacteria but also in Neurospora crassa, aquatic vertebrates (9), and Arabidopsis (AtCRY3) (10). Their putative role as sensory receptors has recently been challenged by showing that they act as repair enzymes for single-stranded DNA (11).The 500 N-terminal amino acids constitute the photolyase homology region (PHR) with reference to the DNA repairing enzyme. In this domain all cryptochromes characterized so far carry flavin adenine dinucleotide (FAD) as light-sensitive chromophore (2, 1...

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Section: Discussioncontrasting

confidence: 48%

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Immeln

Schlesinger

Heberle

et al. 2007

Journal of Biological Chemistry

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125

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“…Because the ribityl side chain and phosphate moiety point toward the domain surface, the adenine moiety must be entirely solvent exposed and is probably highly flexible. This observation explains numerous biochemical findings, such as the instability of FAD bound to BLUF domains and the module's tolerance in binding diverse flavin derivatives (28). In particular, it explains the fact that the nature of the flavin chromophore hardly affects the capacity of the AppA-BLUF domain to photocycle (28,29).…”

Section: Bluf Domains Represent a Previously Uncharacterized Fad Bindingmentioning

confidence: 67%

Structure of a bacterial BLUF photoreceptor: Insights into blue light-mediated signal transduction

Jung

Domratcheva

Tarutina

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

164

278

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Light is an essential environmental factor, and many species have evolved the capability to respond to it. Blue light is perceived through three flavin-containing photoreceptor families: cryptochromes, light-oxygen-voltage, and BLUF (sensor of blue light using flavin adenine dinucleotide, FAD) domain proteins. BLUF domains are present in various proteins from Bacteria and lower Eukarya. They are fully modular and can relay signals to structurally and functionally diverse output units, most of which are implicated in nucleotide metabolism. We present the high resolution crystal structure of the dark resting state of BlrB, a short BLUF domain-containing protein from Rhodobacter sphaeroides. The structure reveals a previously uncharacterized FAD-binding fold. Along with other lines of evidence, it suggests mechanistic aspects for the photocycle that is characterized by a red-shifted absorbance of the flavin. The isoalloxazine ring of FAD binds in a cleft between two helices, whereas the adenine ring points into the solvent. We propose that the adenine ring serves as a hook mediating the interaction with its effector͞output domain. The structure suggests a unique photochemical signaling switch in which the absorption of light induces a structural change in the rim surrounding the hook, thereby changing the protein interface between BLUF and the output domain.blue light sensing ͉ photochemistry ͉ protein function ͉ flavin ͉ reaction mechanism

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“…AppA 5-125 mutants were expressed and purified essentially as described previously (40). Before proceeding with the nickel-resin purification, the cell-free extracts were incubated for 1 h on ice with a large molar excess of FAD.…”

Section: Methodsmentioning

confidence: 99%

“…Purity of the samples was checked by SDS-PAGE using PHASTSystem (Amersham Biosciences) and UV-vis spectroscopy. The flavin composition of each variant was determined by thin-layer chromatography (TLC) as described earlier (40).…”

Section: Methodsmentioning

confidence: 99%

On the Role of Aromatic Side Chains in the Photoactivation of BLUF Domains

et al. 2007

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BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, is investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH • -W • radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electrontransfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.

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Chromophore composition of a heterologously expressed BLUF-domain

Cited by 59 publications

References 20 publications

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Structure of a bacterial BLUF photoreceptor: Insights into blue light-mediated signal transduction

On the Role of Aromatic Side Chains in the Photoactivation of BLUF Domains

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