Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.
Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering.
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DOI：
10.1021/jacs.6b03565
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发表时间：
2016-07-20
影响因子：
15
通讯作者：
Anfinrud PA
中科院分区：
文献类型：
--
作者：
Cho HS;Schotte F;Dashdorj N;Kyndt J;Henning R;Anfinrud PA
应对环境变化的能力对于生物体的生存至关重要。嗜盐盐螺菌 (Halorhodospira halophila) 是一种光合细菌，它会游走远离蓝光，可能是为了逃避能量足以对遗传有害的光子。负责这种反应的蛋白质被认为是光敏黄色蛋白（PYP），其发色团在蓝光存在下从反式光异构化为顺式。我们通过在 100 皮秒到 1 秒的 10 年时间跨度内获取时间分辨小广角 X 射线散射图 (SAXS/WAXS)，研究了完整的 PYP 光循环。使用顺序模型，对时间依赖性散射差异的全局分析恢复了四种中间体（pR0/pR1、pR2、pB0、pB1），其中前三个可以分配给先前的时间分辨晶体结构。 1.8 ms pB0 到 pB1 的转变产生 PYP 信号状态，其回转半径 (Rg = 16.6 Å) 明显大于基态 (Rg = 14.7 Å)，因此时间分辨蛋白质晶体学无法访问。使用 GASBOR 重建的信号状态的形状是高度各向异性的，并且需要蛋白质长轴的显着伸长。这种结构变化与 25 个残基 N 端结构域的展开一致，从而将该感觉蛋白的 β 支架暴露给潜在的结合伴侣。通过时间分辨晶体和溶液研究，对完整 PYP 光循环的机械详细描述成为可能，为理解蛋白质中的信号转导以及评估和验证蛋白质生物物理学中的理论/计算方法提供了一个框架。
The capacity to respond to environmental changes is crucial to an organism’s survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 Å) is significantly larger than that for the ground state (Rg = 14.7 Å) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the β-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.