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

通过红外量子级联激光器与流动池系统的结合来阐明光受体和酶中的不可逆反应

基本信息

批准号：
317120756
负责人：
Professor Dr. Tilman Kottke
金额：
--
依托单位：
Arbeitsgruppe Biophysikalische Chemie und Diagnostik
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/317120756?language=en
关键词：
Elucidation Irreversible Reactions Light Receptors

项目摘要

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.

蛋白质在水中的时间分辨红外光谱分辨率优于毫秒，目前具有挑战性，因为需要非常大的样本量或高效的反应。我们将使用量子级联激光器作为先进的探测光源，具有高发射功率和宽调谐范围，以监测单个波数的反应时间轨迹。将激光的小焦区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率和低样品消耗获得蛋白质中的不可逆光反应。与步进扫描光谱学相比，新装置的性能将在组装后得到彻底的表征。以细菌视紫红质和游离黄素的光反应为模型反应，可以达到时间分辨率和噪声水平的极限。这一方法将被用来阐明两个隐花红光受体和一个同源的DNA修复酶--(6-4)光解酶的机制。隐花色素调节植物的发育，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。作为直接比较，最近发现的类似动物的隐色素aCRY的特征是它对红光的显著反应。此外，还将利用aCRY在UVA光消耗下对DNA(6-4)损伤的修复活性来阐明修复机制中的后期步骤。同源黄素蛋白在三种不同氧化状态下的这些反应都有一个共同点，那就是它们是不可逆的和无效的。因此，以前还没有用时间分辨红外光谱来解决这些问题。这一综合研究将极大地提高我们对隐花色素信号通路和(6-4)光解酶修复机制的理解。特别是，将根据引起构象变化的电荷形成的概念与刚性氧化还原级联的模型来评估不同隐色素中的信号级数。