Time-Resolved Fourier Transform Infrared Spectroscopy on Plant Cryptochrome

隐花色素是一种感觉蛋白，在生物体对蓝光的反应中发挥关键作用。它们设定昆虫和植物的生物钟，并启动植物的发育。此外，在昆虫和候鸟身上，它们还充当地球磁场的传感器。它们将氧化的黄素腺嘌呤二核苷酸作为发色团，维生素B2的衍生物。蓝光的吸收在蛋白质内部引发了一系列事件，其中大部分还没有解决。作为本项目的模型系统，我们选择了绿藻衣藻隐花色素的光敏结构域。植物隐花色素的信号状态是由一个电子和一个质子分别从脱辅基蛋白转移到黄素上而形成的。我们的目标是通过应用傅里叶变换红外光谱，以时间分辨的方式研究蛋白质在纳秒激光激发后的响应。为此，需要调整步进扫描方法以适应隐花色素系统的具体要求。将监测和分析载脂蛋白对微秒质子转移的反应。光反应中的关键中间步骤将在微秒和毫秒的时间尺度上进行解析。分析将揭示质子转移、二级结构变化和氧化还原耦合在多大程度上对信号从生色团一路传递到相互作用伙伴起关键作用。

Cryptochromes are sensory proteins that act as key players in the regulation of organisms in response to blue light. They set the biological clock in insects and plants and initiate plant development. Furthermore, they act as sensors for the Earth’s magnetic field in insects and putatively in migratory birds. They incorporate oxidized flavin adenine dinucleotide as a chromophore, a derivative of vitamin B2. Absorption of blue light initiates a cascade of events within the protein, most of which have not been resolved yet. As a model system for this project, the photosensitive domain of the plant cryptochrome from the green alga Chlamydomonas is selected. The signalling state of plant cryptochromes is formed by separate transfer of an electron and a proton from the apoprotein to the flavin. We aim at investigating the response of the protein in a time-resolved manner by applying Fourier transform infrared spectroscopy after nanosecond laser excitation. For this purpose, the stepscan methodology needs to be adapted to the specific demands of the cryptochrome system. The response of the apoprotein to the microsecond proton transfer will be monitored and analyzed. Crucial intermediate steps in the photoreaction will be resolved on microsecond and millisecond time scales. The analysis will reveal to which extent proton transfer, secondary structural changes, and redox coupling play a key role in transmission of the signal from the chromophore all the way to the interaction partner.