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

隐色素是一种存在于所有生物王国的黄素蛋白，它在昆虫中起着磁感受器的作用，并使植物的生物钟与外界的光暗循环同步。它们在进化上与dna修复光解酶有关。与这些酶不同，隐色素很少结合额外的光收集色素，即所谓的天线发色团，以及光反应性黄素发色团（FAD）。在光解酶中，从天线色素到FAD的能量传递过程已经被深入研究，而在隐色素中，对这些发色团的功能和特性知之甚少。因此，本研究的目的是揭示隐色素中不同天线发色团的超快能量传递过程的差异和共性，以揭示它们与光响应的相关性。来自莱茵衣藻（Chlamydomonas reinhardtii）的类似动物的隐色素aCRY是已知的唯一一种不仅对蓝光有反应，而且对黄光和红光也有反应的隐色素。迄今为止，仅使用携带FAD的aCRY来研究aCRY的红光响应。然而，研究表明，天线发色团8-羟基去氮黄素（8-HDF）与aCRY结合。因此，我想利用超快二维电子能谱来研究携带两个发色团的蛋白质的光响应，以分析发色团之间能量传递的特征和动力学。此外，超快二维电子振动光谱可以在结构水平上阐明天线发色团对aCRY光反应的影响，以及它在蛋白质功能中的作用。为了进行比较，将绿藻类莱茵衣藻（Chlamydomonas reinhardtii）的DASH隐色素CRY-DASH1纳入分析。与aCRY相比，ry - dash1结合了天线5,10-甲基四氢叶酸（MTHF）和黄素发色团。利用二维电子能谱对CRY-DASH1中的能量传递过程进行研究，有助于解决8-HDF和MTHF在能量传递动力学水平上的共性和差异。利用二维电子-振动光谱对crry - dash1中的光响应进行研究，可以将crry - dash1中的结构变化与aCRY中观察到的过程进行比较。因此，所提出的实验可能会阐明不同隐色素中不同天线发色团的相关性。

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.