Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy
Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy
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DOI：
10.1038/s41598-020-59073-5
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发表时间：
2020-02-06
影响因子：
4.6
通讯作者：
Lukacs, Andras
中科院分区：
文献类型：
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作者：
Karadi, Kristof;Kapetanaki, Sofia M.;Lukacs, Andras
使用黄素的蓝光（BLUF）结构域越来越多地被用于光遗传学构建体。尽管如此，关于其激活机制仍有许多问题有待解决。非天然氨基酸突变的出现为蛋白质结构动力学的研究开辟了一个新的工具箱。色氨酸类似物，7-aza-Trp（7AW）被纳入光色素和pucA（AppA）感光体的激活的BLUF结构域，以研究关键的W104残基在蛋白质的光活化过程中的功能动力学。对Trp的7-氮杂修饰使得使用310 nm激发和380 nm发射的选择性激发成为可能，从而将感兴趣的信号与其他Trp和Tyr残基分离。我们使用7AW和黄素之间的福斯特能量转移（FRET）来估计溶液中光适应和暗适应状态下色氨酸和黄素之间的距离。黄素的纳秒荧光各向异性衰减和皮秒荧光寿命测量揭示了色氨酸残基的动态图像。在暗适应状态下，W104的主要群体远离黄素，可以自由移动，与文献中报道的结果相反。在蓝光激发下，占主导地位的色氨酸群体被重组，更接近黄素，占据刚性结合状态，参与黄素分子周围的氢键网络。
Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Forster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.