Lhcx proteins in centric diatoms

硅藻是光合生物，占地球上每年生物量生产的25%左右。它们在类囊体膜中具有不同的蛋白质，用于捕光，由于它们的主要色素而被称为岩藻黄质叶绿素结合蛋白（FCP）。基于序列比较，这些蛋白分为三组，Lhcf蛋白作为主要的光收集器，Lhcr蛋白与光系统I，和Lhcx蛋白。Lhcx通过增加热耗散参与保护免受过量光的影响，这是以称为非光化学猝灭（NPQ）的机制的荧光发射为代价的。NPQ的所谓能量依赖性qE部分取决于类囊体pH梯度、硅藻黄质到硅藻黄质的脱环氧化以及Lhcx蛋白。最近，我们可以证明存在的Lhcx蛋白的三聚体FCPa复合物中的一个中心的硅藻，我们可以表明，这些复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值。然而，硅藻拥有一个以上的Lhcx。因此，我们想研究中心硅藻海链藻中不同的Lhcx蛋白，因为基因组是测序的，而且它是可转化的。首先，我们想要从野生型中纯化FCPa复合物的亚群，并通过质谱法鉴定存在的Lhcx蛋白。在鉴定了存在于不同FCPa亚群中的那些Lhcx之后，我们想要产生T.携带具有相应Lhcx蛋白的标记版本的额外基因的Lhcx蛋白。含有这些Lhcx的特异性FCPa复合物应通过亲和色谱法从转化体中以纯态分离，并进行生物化学表征。然后将在对NPQ重要的条件下，如不同的蛋白质距离、pH值或硅藻黄质含量，用光谱法检查所获得的复合物的荧光发射。此外，将创建相同Lhcx的敲低突变体，并通过荧光动力学研究其NPQ能力，并分析其色素组成。结合体内和体外数据，我们将能够确定NPQ中不同Lhcx蛋白的功能。

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.