Center for Time-Resolved Diffraction, Department of Chemistry, Graduate School of Nanoscience & Technology (WCU), KAIST, Daejeon, 305-701, Republic of Korea.
J Am Chem Soc. 2012 Feb 15;134(6):3145-53. doi: 10.1021/ja210435n. Epub 2012 Feb 3.
Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.
感光蛋白在接收光刺激方面发挥着至关重要的作用，这些光刺激引发了生物功能所需的反应。然而，即使对于原型感光蛋白——光激活黄色蛋白（PYP），其在天然水相环境中的构象转变的结构特征仍然难以捉摸。我们采用泵浦探测 X 射线溶液散射技术，在从 3.16 μs 到 300 ms 的广泛时间范围内探测 PYP 光循环过程中发生的结构变化。通过对中间产物的动力学和结构进行分析，生动地可视化了蛋白质在溶液相中的结构演变过程。我们确定了四个结构上不同的中间产物及其相关的五个时间常数，并从与时间无关的、与物种相关的差散射曲线重建了这四个中间产物的分子形状。中间产物的重建结构显示出较大的构象变化，例如 N 端的突出，由于晶体接触，这种突出在晶体相中受到限制，因此无法通过 X 射线晶体学清楚地观察到。N 端的突出和蛋白质体积随着光循环的进行逐渐增加，在最终的中间产物中达到最大值，该中间产物被认为是信号状态。这些数据不仅揭示了一种普遍的动力学机制适用于晶体相和溶液相，还为样品环境如何影响 PYP 光循环的结构动力学和反应速率提供了直接证据。
