University of Jyväskylä, Nanoscience Center, Department of Biological and Environmental Science, 40014 Jyväskylä, Finland.
Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
Phys Chem Chem Phys. 2021 Mar 11;23(9):5615-5628. doi: 10.1039/d0cp06553f.
Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels.
光感受器蛋白中的信号传递是一个复杂且多维的事件。实时特异性地揭示这些机制是一个可行但具有挑战性的目标。在这里，我们使用非天然氨基酸叠氮苯丙氨酸来阐明红光感应光敏色素中的特异性事件，该氨基酸在振动上可与所有其他蛋白质信号区分开来。在典型的光敏色素中，信号转导始于受激双氢卟啉发色团的异构化，从而启动蛋白质中光感受器单元中的多种过程，最终控制输出域的生化活性，而输出域与发色团相隔纳米级。通过在主要蛋白质位置实施标记，并对 Deinococcus radiodurans 细菌光敏色素进行双色分步扫描傅里叶变换红外光谱分析，我们跟踪了光感受器单元中三个特定位置的信号传递。我们表明，一种结构可切换的发夹延伸结构，即所谓的“舌区”，在微秒内对光转化做出响应，并与发色团同时完成其结构变化，而在毫秒内完成。相比之下，来自其他两个标记位置的动力学表明，即使标记位于发色团附近，特异性变化也偏离了发色团的作用。对其他几个标记位置进行标记会导致光开关性能受损、结构稳定性降低或差光谱中没有变化，这为光感受器单元的内部动力学提供了更多信息。我们的工作阐明了蛋白质在作用下的结构变化的多维性。该研究还表明，通过叠氮探针可以在时间分辨和特异性的方法中研究光敏色素的信号转导机制，并展示了使用这些标记的挑战。
