Analysis of ultrafast energy transfer processes in cryptochromes using transient two-dimensional spectroscopy techniques

隐花色素是存在于所有生命王国中的黄素蛋白，其在昆虫中起磁感受器的作用，并使植物的生物钟与外部光暗周期同步。它们在进化上与DNA修复光裂合酶有关。与这些酶不同，隐花色素很少结合额外的捕光色素，即所谓的天线发色团，以及光反应性黄素发色团（FAD）。在光解的能量转移过程中，从天线色素的FAD已经被彻底研究，而在隐花色素只有很少的是知道这些发色团的功能和特性。因此，拟议的研究项目的目的是揭示隐花色素中不同天线发色团的超快能量转移过程的差异和共性，以揭示它们与光响应的相关性。来自绿色衣藻的类动物隐色素aCRY是唯一已知的不仅对蓝光而且对黄光和红光有反应的隐色素。到目前为止，使用仅携带FAD的aCRY研究了aCRY的红光响应。然而，已经显示天线发色团8-羟基脱氮黄素（8-HDF）与aCRY结合。因此，我想研究的光响应的蛋白质携带两个发色团使用超快二维电子光谱，以分析发色团之间的能量转移的特性和动力学。此外，超快二维电子振动光谱可能阐明天线生色团在结构水平上对aCRY中光反应的影响以及其对蛋白质功能的作用。为了进行比较，将来自绿色莱茵衣藻的DASH隐花色素BASH-DASH 1纳入分析。与aCRY相反，除了黄素发色团之外，EASH-DASH 1还结合天线5，10-亚甲基四氢叶酸（MTHF）。用二维电子光谱研究8-HDF和MTHF之间的能量传递过程可能有助于解决8-HDF和MTHF之间能量传递动力学水平上的共性和差异。用二维电子振动光谱研究DASH 1中的光响应，可以将DASH 1中的结构变化与aCRY中观察到的过程进行比较。因此，所提出的实验可能阐明不同隐花色素中不同触角发色团的相关性。

Cryptochromes are flavoproteins present in all kingdoms of life, which function as magnetoreceptors in insects and synchronize the circadian clock of plants with the external light-dark cycle. They are evolutionary related to DNA-repairing photolyases. Unlike these enzymes, cryptochromes only rarely bind an additional light harvesting pigment, a so called antenna chromophore, alongside the photoreactive flavin chromophore (FAD). In photolyases the energy transfer processes from the antenna pigment to the FAD have already been investigated thoroughly whereas in cryptochromes only little is known about the functionality and characteristics of these chromophores. Consequently, the aim of the proposed research project is to reveal differences and commonalities of the ultrafast energy transfer processes of different antenna chromophores in cryptochromes in order to disclose their relevance for the light response. The animal-like cryptochrome aCRY from the green alga Chlamydomonas reinhardtii is the only known cryptochrome not only responsive to blue but also to yellow and red light. So far, the red light response of the aCRY was investigated using aCRY only carrying FAD. It has been shown, however, that the antenna chromophore 8-hydroxydeazaflavin (8-HDF) binds to aCRY. Therefore, I would like to investigate the light response of the protein carrying both chromophores using ultrafast two-dimensional electronic spectroscopy in order to analyze the characteristics and dynamics of the energy transfer between the chromophores. Additionally, ultrafast two-dimensional electronic-vibrational spectroscopy might elucidate the influence of the antenna chromophore on the photoreaction in aCRY on a structural level as well as its role for the functionality of the protein in general. For comparison, the DASH cryptochrome CRY-DASH1 from the green alga Chlamydomonas reinhardtii will be included into the analysis. In contrast to aCRY, CRY-DASH1 binds the antenna 5,10-methenyltetrahydrofolate (MTHF) in addition to the flavin chromophore. The investigation of the energy transfer processes in CRY-DASH1 with two-dimensional electronic spectroscopy might help to resolve commonalities and differences on the level of energy transfer dynamics between 8-HDF and MTHF. The investigation of the light response in CRY-DASH1 with two-dimensional electronic-vibrational spectroscopy might allow the comparison of structural changes in CRY-DASH1 to the processes observed in aCRY. Therefore, the proposed experiments might elucidate the relevance of different antenna chromophores in different cryptochromes.