Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study
Arguments for an additional long-lived intermediate in the photocycle of the full-length aureochrome 1c receptor: A time-resolved small-angle X-ray scattering study
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DOI：
10.1063/1.5095063
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发表时间：
2019-05-01
影响因子：
2.8
通讯作者：
Kottke, Tilman
中科院分区：
文献类型：
--
作者：
Bannister, Saskia;Boehm, Elena;Kottke, Tilman
金色素（AUREO）在藻类中充当蓝光感受器。它们由光、氧、电压敏感 (LOV) 结构域和 DNA 结合碱性区域/亮氨酸拉链组成。 LOV 中黄素辅因子的照射导致加合物的形成，随后发生整体结构变化。在这里，我们首先应用紫外/可见光谱来表征来自硅藻三角褐指藻的全长金色素 1c (PtAUREO1c) 的光循环。 PtAUREO1c 的时间常数为 850 s，量子产率为 23%，与 PtAUREO1a 相比，其恢复时间更快，对光的灵敏度低得多，这表明它可以作为体内强光传感器。紫外/可见光谱提供了黄素发色团局部恢复的详细信息。然而，全长 AUREO 或任何其他多结构域 LOV 蛋白的整体结构恢复的动力学信息缺失。该信息对于光感受器作为光遗传学设备的应用至关重要。因此，我们建立了一个程序，利用内部装置以时间分辨方式在 PtAUREO1c 上应用小角度 X 射线散射。结合类似条件下的紫外/可见光谱，我们揭示了整体蛋白质结构的恢复和加合物寿命之间的差异。因此，我们建议通过中间态（I447）来补充光循环，该中间态以约800秒的时间常数衰减并延长信号状态的寿命。
Aureochromes (AUREO) act as blue-light photoreceptors in algae. They consist of a light-, oxygen-, voltage-sensitive (LOV) domain and a DNA-binding basic region/leucine zipper. Illumination of the flavin cofactor in LOV leads to the formation of an adduct, followed by global structural changes. Here, we first applied UV/vis spectroscopy to characterize the photocycle of full-length aureochrome 1c (PtAUREO1c) from the diatom Phaeodactylum tricornutum. With a time constant of 850 s and a quantum yield of 23%, PtAUREO1c reveals a faster recovery time and a much lower sensitivity toward light than PtAUREO1a, pointing to its role as a high light sensor in vivo. UV/vis spectroscopy offers details on the local recovery of the flavin chromophore. However, kinetic information on the global structural recovery of full-length AUREO or any other multidomain LOV protein is missing. This information is essential not least for the photoreceptors' applications as optogenetic devices. Therefore, we established a procedure to apply small-angle X-ray scattering on PtAUREO1c in a time-resolved manner employing an in-house setup. In combination with UV/vis spectroscopy under similar conditions, we revealed a discrepancy between the recovery of the global protein structure and the adduct lifetime. Accordingly, we propose to supplement the photocycle by an intermediate state (I447), which decays with a time constant of about 800 s and prolongs the lifetime of the signaling state.