Lhcx proteins in centric diatoms

硅藻是一种光合生物，占地球年生物量产量的25%左右。它们在类囊体膜中具有不同的蛋白质，用于光收集，称为岩藻黄素-叶绿素结合蛋白（FCP），因为它们的主要色素。根据序列比较，这些蛋白可分为三组：Lhcf蛋白作为主要的光收集器，Lhcr蛋白和光系统I相关，Lhcx蛋白。Lhcx通过一种称为非光化学猝灭（NPQ）的机制，以牺牲荧光发射为代价，通过增加散热来防止光过剩。NPQ中所谓的能量依赖qE部分取决于类囊体pH梯度、二氧芑质到二氧芑质的去环氧化以及Lhcx蛋白。最近，我们可以证明在中心硅藻的三聚FCPa配合物中存在一种Lhcx蛋白，并且我们可以证明这些配合物的荧光产率取决于蛋白质-蛋白质的距离，结合的硅藻黄质的量以及ph。然而，硅藻具有不止一种Lhcx。因此，我们想研究不同的Lhcx蛋白在中心硅藻假海藻，因为基因组测序和藻类是可转化的。首先，我们想从野生型中纯化FCPa复合物的亚群，并通过质谱法鉴定存在的Lhcx蛋白。在确定了存在于不同FCPa亚群中的Lhcx之后，我们想要创建假单胞菌的转化体，该转化体携带额外的基因，并带有相应Lhcx蛋白的标记版本。从转化体中分离出含有这些Lhcx的特定FCPa复合物，并通过亲和层析进行纯化，并进行生化表征。然后，在对NPQ重要的条件下，如不同的蛋白质距离、pH值或硅黄素含量，对获得的配合物的荧光发射进行光谱检查。此外，将创建相同Lhcx的敲除突变体，并通过荧光动力学研究其NPQ能力，并分析其色素组成。综合体内和体外数据，我们将能够确定不同Lhcx蛋白在NPQ中的功能。

Diatoms are photosynthetic organisms that contribute to about 25% to the yearly biomass production on earth. They possess different proteins in the thylakoid membrane working in light harvesting, called fucoxanthin-chlorophyll-binding proteins (FCP) because of their major pigments. Based on sequence comparison those fall into three groups, Lhcf proteins serving as main light harvesters, Lhcr proteins associated with photosystem I, and Lhcx proteins. Lhcx are involved in protection against a sur-plus of light by increased heat dissipation at the expense of fluorescence emission by a mechanism called non-photochemical quenching (NPQ). The so-called energy dependent qE part of NPQ depends on the transthylakoidal pH gradient, the de-epoxidation of diadinoxanthin to diatoxanthin and on the Lhcx proteins. Recently, we could prove the existence of one of the Lhcx proteins in trimeric FCPa complexes in a centric diatom, and we could show that the fluorescence yield of these complexes depends on the protein-protein distance, the amounts of diatoxanthin bound as well as on the pH. However, diatoms possess more than one Lhcx. Thus we want to study the different Lhcx proteins in the centric diatom Thalassiosira pseudonana, since the genome is sequenced and the alga is transformable. First we want to purify sub-populations of the FCPa complexes from wild type and identify the Lhcx proteins present by mass spectrometry. Having identified those Lhcx present in the different FCPa subpopulations, we want to create transformands of T. pseudonana carrying additional genes with tagged versions of the respective Lhcx proteins. From the transformands the specific FCPa complexes containing these Lhcx should be isolated in a pure state by affinity chromatography, and characterised biochemically. Complexes obtained will then be examined spectroscopically for their fluorescence emission under conditions important for NPQ, like varying protein distances, pH or diatoxanthin content. In addition, knock-down mutants for the same Lhcx will be created and studied for their NPQ capacity by fluorescence kinetics, and their pigment composition will be analysed. Taking together the in vivo and in vitro data we will be able to determine the function of the different Lhcx proteins in NPQ.