Functional characterisation of different fucoxanthin chlorophyll proteins (FCP) in diatoms

本项目的目的是了解硅藻、岩藻黄质-叶绿素蛋白(FCPS)天线复合体中的激发能量传递，FCPS可能的保护作用，以及FCP蛋白在光系统周围的大分子组织。尽管可以在基因水平上区分几组FCP，但通常只纯化了一到两个主要的天线复合体，关于不同FCP的生化和光谱数据很少。因此，人们对这些不同多肽可能的功能异质性知之甚少。为了表征单天线蛋白质，我们想要转化带有不同FCP基因的硅藻，通过添加标签来修饰，以便能够根据所有相关蛋白质的背景分离特定的FCP。此外，可能的色素结合部位将发生突变，以获得不同色素沉着的FCP。然后，基因工程的FCP应该允许进行详细的生化和光谱表征。根据其在不同FCP中的定位，将特别强调双黄素在荧光猝灭中的作用。在研究突变体FCP的同时，还将研究不同的FCP蛋白在不同的光系统中的排列。此外，使用组氨酸标记来标记FCP用于电子显微镜将允许在光系统的超复合体中特定地定位FCP。

The aim of this project is to understand the excitation energy transport in the antenna complexes of diatoms, fucoxanthin-chlorophyll proteins (FCPs), the possible protective function of FCPs and the macromolecular organisation of the FCP proteins around the photosystems. Despite the fact that several groups of FCPs can be distinguished on the level of genes, usually only one or two main antenna complexes are purified and biochemical and spectroscopic data on distinct FCPs are scarce. Thus, little is known about possible functional heterogeneities of these different polypeptides. In order to characterise single antenna proteins we want to transform diatoms with different FCP genes modified by addition of tags in order to be able to separate specific FCPs against the background of all related proteins. Additionally, putative pigment binding sites will be mutated to obtain FCPs with different pigmentation. The genetically engineered FCPs should then allow for a detailed biochemical and spectroscopic characterisation. Special emphasis will be on the role of diatoxanthin in fluorescence quenching depending on its localisation in the different FCPs. The studies on mutant FCPs will be accompanied by studies on the arrangement of the different FCP proteins with respect to the different photosystems. Additionally, using the His-tags to label FCPs for electron microscopy will allow to specifically locate the FCPs inside supercomplexes of the photosystems.