A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis

Iseki, Mineo; Matsunaga, Shigeru; Murakami, Akio; Ohno, Kaoru; Shiga, Kiyoshi; Yoshida, Kanako; Sugai, Manabu; Takahashi, Tetsuo; Hori, Takafumi; Watanabe, Masakatsu

doi:10.1038/4151047a

Cited by 451 publications

(420 citation statements)

References 26 publications

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“…Such heterogeneity of the ground state was previously observed in wild-type Slr1694 (19,22), Thermococcus elongatus TePixD (24), and AppA and its mutants (9,20,35). The absence of an obvious H/D exchange effect on the decay of FAD* [1][2][3][4] indicates that FAD* is deactivated through electron transfer, as for wild-type Slr1694 (19, 21, 22). The decay of FAD* is highly multiexponential and distributed between a few picoseconds and ∼250 ps in wild-type Slr1694 (19,21,22) and the W91F mutant, which is assigned to different conformational subpopulations of the tyrosine side chain having variations in the distance to FAD, with an ensuing distribution of electron transfer rates (9,20).…”

Section: Resultsmentioning

confidence: 59%

“…Especially members of the BLUF 1 (blue light photoreceptors using FAD) family (1)(2)(3) show an especially intriguing light-induced proton network rearrangement resulting in a 10-15 nm red-shifted spectrum of the signaling state. The BLUF domain shows a ferredoxin-like fold consisting of a five-stranded β-sheet with two R-helices packed on one side of the sheet, with the isoalloxazine ring of flavin adenine dinucleotide (FAD) positioned between the two R-helices (4)(5)(6)(7)(8)(9)(10)(11)(12).…”

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confidence: 99%

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The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

et al. 2009

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BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH • that was formed in ∼1 ps and recombines in 10 ps and is tentatively assigned to a FADH • -W8 • radical pair. The second deactivation branch forms FADH • in 8 ps and evolves to FAD •-in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.Blue light photoreceptors using flavin cofactors have been the focus of recent research because of their novel mechanisms of photoactivation in contrast to "classical" photoreceptors like phytochromes and rhodopsins. Especially members of the BLUF 1 (blue light photoreceptors using FAD) family (1-3) show an especially intriguing light-induced proton network rearrangement resulting in a 10-15 nm red-shifted spectrum of the signaling state. The BLUF domain shows a ferredoxin-like fold consisting of a five-stranded β-sheet with two R-helices packed on one side of the sheet, with the isoalloxazine ring of flavin adenine dinucleotide (FAD) positioned between the two R-helices (4-12). FAD is noncovalently bound to the protein through a number of hydrogen bonds and hydrophobic interactions. Figure 1 shows the three-dimensional structure of the Synechocystis Slr1694 BLUF domain (also known as PixD) in its proposed dark and light states, with the FAD binding pock...

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Section: Resultsmentioning

confidence: 59%

mentioning

confidence: 99%

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

et al. 2009

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“…than the activity obtained in a previous study 7 may be related to the modification in the purification steps. To elucidate the fluence rate-response relationships in PAC activation, we measured the activity of PAC under blue light at various photon fluence rates.…”

Section: Dependency On Photon Fluence Ratesmentioning

confidence: 53%

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena

Yoshikawa

Suzuki

Watanabe

et al. 2005

Photochem Photobiol Sci

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Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2-50 lmol m −2 s −1 (total fluence of 1200 lmol m −2 ). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.

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“…The novelty of this device for optical biosensing is that the MLA enables in parallel detecting and counting cells with coupling-free white light. For the biosamples to be detected, 60 cells of Euglena, which is a popular flagellated laboratory microorganism living in freshwater environments and representing one of the simplest and earliest derived eukaryotic cells, 36 were introduced. The optofluidics recorded 56 intensity drops, corresponding to a 93% success rate for detection.…”

Section: D Designable Optofluidic Devices D Wu Et Almentioning

confidence: 99%

In-channel integration of designable microoptical devices using flat scaffold-supported femtosecond-laser microfabrication for coupling-free optofluidic cell counting

Niu

et al. 2015

Light Sci Appl

118

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The high-precision integration of three-dimensional (3D) microoptical components into microfluidics in a customizable manner is crucial for optical sensing, fluorescence analysis, and cell detection in optofluidic applications; however, it remains challenging for current microfabrication technologies. This paper reports the in-channel integration of flexible two-dimensional (2D) and 3D polymer microoptical devices into glass microfluidics by developing a novel technique: flat scaffold-supported hybrid femtosecond laser microfabrication (FSS-HFLM). The scaffold with an optimal thickness of 1-5 mm is fabricated on the lower internal surface of a microfluidic channel to improve the integration of high-precision microoptical devices on the scaffold by eliminating any undulated internal channel surface caused by wet etching. As a proof of demonstration, two types of typical microoptical devices, namely, 2D Fresnel zone plates (FZPs) and 3D refractive microlens arrays (MLAs), are integrated. These devices exhibit multicolor focal spots, elongated (.three times) focal length and imaging of the characters 'RIKEN' in a liquid channel. The resulting optofluidic chips are further used for coupling-free white-light cell counting with a success rate as high as 93%. An optofluidic system with two MLAs and a W-filter is also designed and fabricated for more advanced cell filtering/counting applications.

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A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis

Cited by 451 publications

References 26 publications

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena

In-channel integration of designable microoptical devices using flat scaffold-supported femtosecond-laser microfabrication for coupling-free optofluidic cell counting

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