Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy
Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy
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DOI：
10.1021/acs.jpclett.6b01484
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发表时间：
2016-09-01
影响因子：
5.7
通讯作者：
Kennis, John T. M.
中科院分区：
文献类型：
--
作者：
Konold, Patrick E.;Mathes, Tilo;Kennis, John T. M.
生物光感受器的光触发反应由于其在天然生物体中作为分子开关的作用和光遗传学应用而受到极大的关注。植物趋光素的光、氧和电压2（LOV 2）感测结构域结合C-末端J α螺旋，该螺旋停靠在β-折叠上并且在由黄素单核苷酸（FMN）发色团吸收光时展开。本研究利用时间分辨红外光谱技术从皮秒到微秒的范围研究了燕麦LOV 2的信号转导途径。在D2 O缓冲液中，FMN单重态到三重态的转化在2ns内发生，共价半胱氨酰-FMN加合物在10 μ s内形成。我们观察到一个两步展开的J α螺旋：第一阶段伴随着Cys-FMN共价加合物的形成，在10 μ s，沿着与氢键断裂的FMN C4=O与谷氨酰胺-513，运动的β-片层，和一个额外的螺旋元素。第二阶段发生在大约240 μ s。在500 μ s的最终光谱基本上是相同的稳态光减暗傅立叶变换红外光谱，表明J α螺旋展开是在该时间尺度上完成。
Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal J alpha helix that is docked on a beta-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 mu s. We observe a two-step unfolding of the J alpha helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 mu s, along with hydrogen-bond rupture of the FMN C4=O with Gln-513, motion of the beta-sheet, and an additional helical element. The second phase occurs in approximately 240 mu s. The final spectrum at 500 mu s is essentially identical to the steadystate light-minus-dark Fourier transform infrared spectrum, indicating that J alpha helix unfolding is complete on that time scale.