Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.
Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.
复制标题
DOI：
10.1021/acs.jpcb.7b00088
复制
发表时间：
2017-02-09
期刊：
影响因子：
--
通讯作者：
Meech SR
中科院分区：
文献类型：
--
作者：
Gil AA;Laptenok SP;French JB;Iuliano JN;Lukacs A;Hall CR;Sazanovich IV;Greetham GM;Bacher A;Illarionov B;Fischer M;Tonge PJ;Meech SR
光敏蛋白的合理工程支撑了光遗传学领域，其中光被用于细胞信号的时空控制。光遗传元件的功能是将嵌入式发色团的电子激发转化为微秒到秒时间尺度上的结构变化，然后调节负责生物信号传导的输出域的活动。利用时间分辨振动光谱结合同位素标记技术，我们绘制了黄素结合趋光性蛋白Avena sativa （AsLOV2）的LOV2结构域在10多年时间内的结构演变，报告了光学激发后100飞秒到1毫秒之间的结构动力学。瞬态振动光谱包含黄素发色团和周围蛋白质基质的贡献。这些贡献是通过研究四个不同的同位素标记样品来解决和分配的。高信噪比数据允许详细分析与光激活结构演化相关的动力学。提出了一种与数据一致的光循环途径。最早的事件发生在黄素结合袋中，在那里蛋白质基质发生亚皮秒的扰动。在这种扰动环境中，先前描述的三重态异氧嘧啶和相邻的半胱氨酸之间的反应导致加合状态的形成；这一步骤显示出弥散动力学。该反应促进了光激发与连续的时间相关结构变化的耦合，最初发生在AsLOV2结构域的β-薄片和α-螺旋区域，最终导致j - α-螺旋展开，产生信号状态。该模型通过点诱变进行验证，特别阐明了Q513所起的关键介导作用。
The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatio-temporal control of cell signalling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds timescale, which then modulate the activity of output domains responsible for biological signalling. Using time resolved vibrational spectroscopy coupled with isotope labelling we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 femtoseconds and one millisecond after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labelled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a sub-picosecond perturbation of the protein matrix occurs. In this perturbed environment the previously characterised reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the β-sheet then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signalling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.