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首页> 外文期刊>Biochemistry >Dynamics of Light-Induced Activation in the PAS Domain Proteins LOV2 and PYP Probed by Time-Resolved Tryptophan Fluorescence
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Dynamics of Light-Induced Activation in the PAS Domain Proteins LOV2 and PYP Probed by Time-Resolved Tryptophan Fluorescence

机译：时间分辨色氨酸荧光探测PAS域蛋白LOV2和PYP中光诱导活化的动力学。

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Light-induced activation of the LOV2-JR domain of the photoreceptor phototropin from oat isnbelieved to involve the detachment of the JR helix from the central β-sheet and its subsequent unfolding. Thendynamics of these conformational changes were monitored by time-resolved emission spectroscopy with 100 nsntime resolution. Three transitions were detected during the LOV2-JR photocycle with time constants of 3.4 μs,n500 μs, and 4.3 ms. The fastest transition is due to the decay of the flavin phosphorescence in the transition ofnthe triplet LOVLn660 state to the singlet LOVSn390 signaling state. The 500 μs and 4.3 ms transitions are due tonchanges in tryptophan fluorescence andmay be associated with the dissociation and unfolding of the JR helix,nrespectively. They are absent in the transient absorption signal of the flavin chromophore. The tryptophannfluorescence signal monitors structural changes outside the chromophore binding pocket and indicates thatnthere are at least three LOVSn390 intermediates. Since the 500 μs and 4.3 ms components are absent in anconstruct without the JR helix and in the mutantW557S, the fluorescence signal is mainly due to tryptophann557. The kinetics of the main 500 μs component is strongly temperature dependent with activation energy ofn18.2 kcal/mol suggesting its association with a major structural change. In the structurally related PASndomain protein PYP the N-terminal cap dissociates from the central β-sheet and unfolds upon signaling statenformation with a similar time constant of ∼1 ms. Using transient fluorescence we obtained a nearly identicalnactivation energy of 18.5 kcal/mol for this transition.
机译：人们认为，光诱导的燕麦光感受器光蛋白的LOV2-JR结构域的活化涉及JR螺旋从中心β-折叠层的分离以及随后的展开。然后通过时间分辨发射光谱以100 nsntime的分辨率监测这些构象变化的动力学。在LOV2-JR光周期中检测到三个跃迁，其时间常数分别为3.4μs，n500μs和4.3 ms。最快的跃迁是由于在三重态LOVLn660状态到单重态LOVSn390信号状态跃迁中黄素磷光的衰减。 500μs和4.3 ms的跃迁是色氨酸荧光的tonton变化引起的，可能分别与JR螺旋的解离和展开有关。黄素发色团的瞬态吸收信号中没有它们。色氨酸荧光信号监测生色团结合口袋外部的结构变化，并表明其中至少有三个LOVSn390中间体。由于在没有JR螺旋的构造物中和在突变体W557S中不存在500μs和4.3 ms的分量，因此荧光信号主要归因于色氨酸557。主要的500μs组分的动力学与温度密切相关，活化能为n18.2 kcal / mol，表明其与主要的结构变化有关。在结构相关的PASndomain蛋白PYP中，N端帽从中央β-折叠层解离，并在发出信号状态信息时以约1 ms的相似时间常数展开。使用瞬态荧光，我们为该跃迁获得了几乎相同的18.5 kcal / mol的活化能。

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