Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap.
Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap.
复制标题
染料标记的光敏黄色蛋白的时间分辨光谱表明光诱导 N 端帽结构变化的途径。
DOI：
10.1039/b821345c
复制
发表时间：
2009
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
光感受器 PYP 通过整体构象变化响应光激活。这些变化主要位于蛋白质的 N 端帽，距离发色团结合袋约 20 Å，并通过中央 β 折叠与其分开。 PAS 结构域蛋白超家族普遍关注中心 β-折叠结构变化的传播问题，其中 PYP 是该超家族的结构原型。在这里，我们通过瞬态吸收光谱在纳秒到秒的时间尺度上测量了 N 端帽结构变化的动力学。为此，制备了半胱氨酸突变体 A5C 和 N13C，并用硫醇反应性 5-碘乙酰胺荧光素 (IAF) 进行标记。 A5 位于 N 末端附近，而 N13 是螺旋 α1 的一部分，靠近 N 末端帽和中央反平行 β 片层之间功能重要的盐桥 E12-K110。染料的吸收光谱对其环境敏感，并可作为标记位点附近构象变化的传感器。在两种标记突变体中，光激活导致荧光素吸收光谱短暂红移。为了将构象变化与蛋白质的光循环中间体联系起来，我们将染料的瞬时吸收信号的动力学与对羟基肉桂酰发色团的动力学进行了比较。虽然 A5 附近的结构变化与约 200 μs 内形成的 I2 中间体的上升同步，但 N13 附近的变化被延迟并随着下一个中间体 I2' 的上升而上升，I2' 在约 2 ms 内形成。这表明 N 端帽的不同部分以不同的动力学响应光激活。对于光敏黄色蛋白的信号传导途径，我们提出了一个模型，其中结构信号从发色团结合袋穿过中央β片层，通过N端区域传播到螺旋α1，导致蛋白质构象发生巨大变化。
The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is ∼20 Å away from the chromophore binding pocket and separated from it by the central β-sheet. The question of the propagation of the structural change across the central β-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix α1 near the functionally important salt bridge E12–K110 between the N-terminal cap and the central anti-parallel β-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I2 intermediate, which is formed in ∼200 μs, the change near N13 is delayed and rises with the next intermediate I2′, which forms in ∼2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central β-sheet via the N-terminal region to helix α1, resulting in a large change in the protein conformation.