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品量或高效的反应，目前具有挑战性的是，在H2O中对蛋白质进行时间分辨红外光谱分析，其分辨率优于毫秒。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。激光的小聚焦区域与我们实验室建立的H2O流动池系统相结合，将使我们能够以低产量和低样品消耗获得蛋白质中的不可逆光反应。新装置的性能将在组装后与步进扫描光谱法进行比较。的设置相对于时间分辨率和噪声水平的限制将达到使用的光反应的细菌视紫红质和游离黄素作为模型反应。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）光裂合酶的机制。隐花色素调节植物发育，控制植物和昆虫的日常节律，并作为磁感受器。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，最近发现的动物样隐花色素aCRY将其特征在于其显着的响应红光。此外，将利用UVA光消耗下aCRY对DNA中的（6-4）损伤的修复活性来阐明修复机制中的后期步骤。三种不同氧化态的同源黄素蛋白的这些反应都有一个共同点，即它们是不可逆的和低效的。因此，它们以前没有被解决的时间分辨红外光谱。该研究将有助于加深我们对隐花色素信号通路和（6-4）光裂合酶修复机制的理解。特别是，在不同的隐花色素的信号进展将评估相对于一个刚性的氧化还原级联模型的电荷形成引起构象变化的概念。

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.