Elucidation of Irreversible Reactions in Light Receptors and Enzymes by the Combination of an Infrared Quantum Cascade Laser with a Flow Cell System

由于需要非常大的样本量或高效的反应，对水中蛋白质进行分辨率优于毫秒的时间分辨红外光谱分析目前具有挑战性。我们将采用量子级联激光器作为一种先进的探测光源，具有高发射功率和宽调谐范围，以监测单波数反应的时间轨迹。激光的小聚焦面积与我们实验室建立的水的流动池系统相结合，将使我们能够以低产量和低样品消耗的方式获得蛋白质中的不可逆光反应。与步进扫描光谱相比，组装后的新装置的性能将得到彻底的表征。使用细菌视紫红质和游离黄素的光反应作为模型反应，将达到该装置在时间分辨率和噪声水平方面的极限。该方法将用于阐明两种隐花色素光受体和一种同源DNA修复酶（6-4）光解酶的机制。隐色素调节植物发育，控制植物和昆虫的日常节律，并充当磁感受器。我们将研究在ATP结合的影响下植物隐花色素对蓝光的结构响应。为了直接比较，最近发现的类似动物的隐花色素aCRY将以其对红光的显着反应为特征。此外，在UVA光消耗下，aCRY对DNA（6-4）损伤的修复活性将被用来阐明修复机制的后期步骤。同源黄酮类蛋白在三种不同氧化态下的反应都具有不可逆和低效率的共同特点。因此，这些问题以前还没有被时间分辨红外光谱所解决。这项全面的研究将大大提高我们对隐色素信号通路和（6-4）光解酶修复机制的认识。特别是，不同隐色素的信号进展将根据引起构象变化的电荷形成概念与刚性氧化还原级联模型进行评估。

Time-resolved infrared spectroscopy on proteins in H2O with a resolution better than milliseconds is currently challenging because of the need for very large sample quantities or highly efficient reactions. We will employ a quantum cascade laser as an advanced probe light source with high emission power and broad tuning range to monitor time traces of reactions at single wavenumbers. The combination of the small focal area of the laser with a flow cell system for H2O established in our laboratory will allow us to gain access to irreversible photoreactions in proteins with low yields and with low sample consumption. The performance of the new setup will be thoroughly characterized after assembly in comparison to that of step-scan spectroscopy. The limits of the setup with respect to time resolution and noise level will be reached using the photoreactions of bacteriorhodopsin and free flavin as model reactions. The approach will be utilized to elucidate the mechanisms of two cryptochrome light receptors and a homologous DNA repair enzyme, the (6-4) photolyase. Cryptochromes regulate plant development, govern the daily rhythm of plants and insects, and act as magnetoreceptors. We will study the structural response of a plant cryptochrome to blue light under the influence of ATP binding. For direct comparison, the recently found animal-like cryptochrome aCRY will be characterized with respect to its remarkable response to red light. Furthermore, the repair activity of aCRY on (6-4) lesions in DNA under UVA light consumption will be exploited to elucidate the late steps in the repair mechanism. These reactions of homologous flavoproteins in three different oxidation states all have in common that they are irreversible and inefficient. Therefore, they have not been addressed previously by time-resolved infrared spectroscopy. The comprehensive study will significantly improve our understanding of the signaling pathways of cryptochromes and repair mechanisms of (6-4) photolyases. In particular, signal progression in different cryptochromes will be evaluated with respect to the concept of charge formation causing conformational changes versus the model of a rigid redox cascade.