Distinguishing chromophore structures of photocycle intermediates of the photoreceptor PYP by transient fluorescence and energy transfer.
Distinguishing chromophore structures of photocycle intermediates of the photoreceptor PYP by transient fluorescence and energy transfer.
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通过瞬态荧光和能量转移区分光感受器 PYP 的光循环中间体的发色团结构。
DOI：
10.1021/jp801174z
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Heyn,MaartenP
中科院分区：
文献类型：
--
作者：
Hoersch,Daniel;Otto,Harald;Cusanovich,MichaelA;Heyn,MaartenP
肉桂酰发色团是光感受器光敏黄色蛋白 (PYP) 的光激活开关，并在功能周期中异构化。 W119（PYP 的唯一色氨酸）的荧光通过能量转移到发色团而被猝灭。这取决于发色团的跃迁偶极矩方向和光谱，两者都会在光循环过程中发生变化。因此，W119 的瞬态荧光可作为发色团结构和方向的灵敏动力学监测器，并首次用于研究光循环动力学。根据这些数据和背景照明 (470 nm) 下 ps 荧光衰减的测量结果，我们确定了 I1 和 I1' 中间体中 W119 的荧光寿命。根据时间分辨 X 射线衍射，提出了 I1 光中间体的两种共存的不同发色团结构（Ihee, H., et al. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 7145）：一种与 E46 和 Y42 具有两个氢键，第二种与 Y42 仅有一个氢键且具有不同的方向。仅对于其中第一个，计算的荧光寿命为 0.22 ns，与观测到的 0.26 ns 非常吻合。第二种结构的预计寿命为 0.71 ns。因此，我们得出结论，在解中仅出现第一个 I1 结构。 I1′中间体（I1在碱性pH下的衰变产物）的高分辨率结构仍然未知。我们根据观察到的 1.3 ns 寿命预测，I1' 的发色团结构与 I2 中间体的发色团结构非常相似，因此 I1' 应被视为 I2 的碱性（去质子化）形式。
The cinnamoyl chromophore is the light-activated switch of the photoreceptor photoactive yellow protein (PYP) and isomerizes during the functional cycle. The fluorescence of W119, the only tryptophan of PYP, is quenched by energy transfer to the chromophore. This depends on the chromophore’s transition dipole moment orientation and spectrum, both of which change during the photocycle. The transient fluorescence of W119 thus serves as a sensitive kinetic monitor of the chromophore’s structure and orientation and was used for the first time to investigate the photocycle kinetics. From these data and measurements of the ps-fluorescence decay with background illumination (470 nm) we determined the fluorescence lifetimes of W119 in the I1and I1′ intermediates. Two coexisting distinct chromophore structures were proposed for the I1photointermediate from time-resolved X-ray diffraction (Ihee, H., et al. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 7145): one with two hydrogen bonds to E46 and Y42, and a second with only one H-bond to Y42 and a different orientation. Only for the first of these is the calculated fluorescence lifetime of 0.22 ns in good agreement with the observed one of 0.26 ns. The second structure has a predicted lifetime of 0.71 ns. Thus, we conclude that in solution only the first I1structure occurs. The high resolution structure of the I1′ intermediate, the decay product of I1at alkaline pH, is still unknown. We predict from the observed lifetime of 1.3 ns that the chromophore structure of I1′ is quite similar to that of the I2intermediate, and I1′ should thus be considered as the alkaline (deprotonated) form of I2.