The D2 Protein of Photosystem II Studied in Systems without Photosystem I

D2蛋白是光系统II的两个反应中心蛋白之一，是植物和蓝藻类囊体膜中的一种色素蛋白复合物。这个光系统利用光能氧化水（产生氧气），并产生还原性等价物，用于光合作用的后续步骤。D2蛋白结合一些辅助因子和辅助基团，参与光合作用电子传递。此外，它还含有一种氧化还原活性的三叶草基残留物，在其氧化（自由基）形式下可稳定达数小时。蛋白质环境为辅助因子和辅基提供了合适的取向、定位和氧化还原性质，使光诱导的电子转移反应快速有效。D2蛋白的靶向诱变用于鉴定D2中与辅因子相互作用并影响其功能特征的结构域和残基。将靶向突变引入可转化（照片）异养蓝藻Synechocystis sp. PCC 6803，该细菌通过同源重组将外源DNA整合到其基因组中。这允许删除编码D2蛋白的野生型基因，然后引入一个携带突变的基因拷贝（在基因组的原始位置）。为了避免由于光系统I（一种叶绿素结合蛋白复合物，也显示光诱导电子传递）的存在而导致的功能分析的复杂性，我们构建了一个耐光系统I-less的Synechocystis 6803菌株，它可以作为一个方便的受体菌株引入D2突变。在现有数据的基础上，已经确定了一些非常有趣的D2残基，并且在光系统I-less背景下，这些残基将引入突变。由此产生的突变体将通过多种方法进行分析，无论是在体内还是在类囊体制剂中，包括荧光诱导和发射，电子顺磁共振波谱学和闪光诱导析氧。该实验系统为详细分析由蛋白质环境调节的辅因子特性提供了极好的机会。来自蓝藻的光系统II复合体已被证明是研究蛋白质/辅因子相互作用的合适模型系统，并且耐光系统I-less菌株的开发现在允许在相对完整的系统中进行此类研究。此外，耐光系统I-less synnechocystis菌株为D2蛋白的随机诱变提供了一个非常好的系统，因为它允许对光系统II活性受损的突变体进行正选择。保留外围天线系统的无光系统突变体在野生型10%的光强下繁殖良好，但不能达到全部光强；这似乎是由于在没有光系统i的情况下，细胞无法处理太多光系统ii产生的电子。这一特性可以用来选择随机产生的电子传递受损的D2突变体。在大肠杆菌中携带D2基因的质粒中引入随机突变后，该质粒可用于转化无光系统i和无光系统D2的聚囊菌菌株。在选择转化体后，电子流受损的细胞（由D2突变引起）很容易被耐光表型挑出来。随后，可以确定突变的位置及其结构和功能影响。在光系统I-less背景下研究D2蛋白的提议项目将详细了解各种残基和结构域在促进高效和快速电子转移中的作用。该研究不仅将增强对光系统II的理解，而且将适用于更广泛的蛋白质折叠和稳定性中辅助因子/蛋白质相互作用的视角，并将为相当不寻常的蛋白质氧化还原化学提供信息，例如酪氨酸氧化导致稳定的自由基，正如在D2蛋白中发现的那样。这项工作涉及分子生物学、微生物学、生物化学和某些生物物理学方面，非常适合广泛和跨学科的研究生和博士后培训。光合作用是利用光促进固碳和释氧的过程。基本上，地球上所有的有机物质和氧气都来自光合作用。这个项目解决的问题是光合作用的一个基本步骤是如何工作的。从生物化学和生物物理学的角度来看，这种理解是重要的，并且对生物膜中蛋白质复合物的结构、功能和组装具有一般意义。这一步骤将由一种色素结合蛋白复合物实现，包括将光能非常有效地转化为生物体可以使用的化学能，同时将水转化为氧气。我们将在蓝藻（蓝绿藻）中使用遗传操作来特异性地改变参与该过程的色素结合蛋白。基因操作还将用于从生物体中删除选定的其他色素结合蛋白，以便对正在研究的步骤进行功能分析。该项目将导致(a)加强对色素和蛋白质之间相互作用的理解，这对光合作用至关重要，(b)提高对蛋白质特定残基在催化（增强）特定生化反应中的作用的认识。*** u ~R b C E Ss ' $ - / 1 3 C ' ' $ $ $ $ $ ！$ $ $ E E S E 2 Times New Roman Symbol & Arial & France p- " h .

Vermass 9316857 The D2 protein is one of the two reaction center proteins of photosystem II, a pigment protein complex in the thylakoid membrane of plants and cyanobacteria. This photosystem utilizes light energy to oxidize water (resulting in oxygen) and to produce reducing equivalents to be used in subsequent steps of photosynthesis. The D2 protein binds a number of cofactors and prosthetic groups, which are involved in photosynthetic electron transport. In addition, it contains a redox-active tryosyl residue, which is stable in its oxidized (radical) form for up to several hours. The protein environment provides cofactors and prosthetic groups with the appropriate orientation, localization, and redox properties to make light-induced electron-transfer reactions fast and efficient. Targeted mutagenesis of the D2 protein is applied to identify the domains and residues in D2 that interact with cofactors and affect their functional characteristics. Targeted mutations are introduced into the transformable (photo)heterotrophic cyanobacterium Synechocystis sp. PCC 6803, which incorporates foreign DNA into its genome by homologous recombination. This allows a deletion of the wild-type genes coding for the D2 protein, followed by an introduction of a gene copy (at its native position in the genome) carrying a mutation. To avoid complications in the functional analysis stemming from the presence of photosystem I (a chlorophyll-binding protein complex, which also shows light-induced electron transport), a light-tolerant photosystem I-less strain of Synechocystis 6803 has been constructed, which can be genetically tai lored as a convenient receptor strain to introduce D2 mutations. On the basis of existing data, a number of highly interesting D2 residues have been identified, and mutations will be introduced in these residues in a photosystem I-less background. The resulting mutants will be analyzed, either in vivo or in thylakoid preparations, by a number of methods, including fluorescence induction and emission, electron paramagnetic resonance spectroscopy, and flash-induced oxygen evolution. This experimental system provides an excellent opportunity for detailed analysis of cofactor properties as modulated by the protein environment. The photosystem II complex from cyanobacteria has proven to be a suitable model system to study protein/cofactor interactions, and the development of a light-tolerant photosystem I-less strain now allows such studies to be carried out in relatively intact systems. In addition, the light-tolerant photosystem I-less Synechocystis strain provides a very good system for random mutagenesis of the D2 protein, because it allows for positive selection of mutants with impaired photosystem II activity. Photosystem I-less mutants retaining the peripheral antenna system propagate well at 10% of the light intensity used for wild type, but not a full light intensity; this appears to be caused by an inability of the cell to cope with too many photosystem II-generated electrons in the absence of photosystem I. This property can be take advantage of to select randomly generated D2 mutants with impaired electron transport. After introduction of random mutations in a plasmid carrying the D2 gene in Escherichia coli, this plasmid can be used for transformation of a photosystem I-less and D2-less strain of Synechocystis. After selection for transformants, cells with impaired electron flow (resulting from a D2 mutation) easily can be picked out by a light-resistant phenotype. Subsequently, the site of the mutation and its structural and functional effects can be determined. The propo sed project on the D2 protein studied in a photosystem I-less background will result in detailed insight in the role various residues and domains play in facilitating efficient and rapid electron transfer. The research not only will enhance the understanding of photosystem II, but also will be applicable in a broader perspective of cofactor/protein interactions in protein folding and stability, and will be informative regarding rather unusual protein redox chemistry, such as tyrosyl oxidation leading to a stable radical, as is found in the D2 protein. this work involves molecular biology, microbiology, biochemistry, and certain biophysical aspects, and is very well suited for a broad and interdisciplinary graduate and postdoctoral training. %%% Photosynthesis is the process in which light is utilized to fuel carbon fixation and oxygen evolution. Essentially all organic material and oxygen on earth originates from photosynthesis. The question addressed in this project is how one of the fundamental steps in photosynthesis works. Such understanding is important from both a biochemical and biophysical point of view , and has general implications for structure, function, and assembly of protein complexes in biological membranes. The step that will be addressed is made possible by a pigment-binding protein complex, and involves a very efficient conversion of light energy to chemical energy that can be used by living organisms, with a concomitant conversion of water to oxygen. We will use genetic manipulation in a cyanobacterium (blue-green alga) to specifically change pigment-binding proteins involved in this process. Genetic manipulation also will be utilized to delete selected other pigment-binding proteins from the organism, so that functional analysis of the steps being studied is greatly facilitated. This project will result in (a) enhanced understanding of the interplay between pigments and proteins that is crucial for photosynthesis, and (b) improved insight in the role specific r esidues of proteins play in catalysis (enhancement) of particular biochemical reactions. *** u ~ R b C E S s ı $ - / 1 3 C E ı $ $ $ $ $ ! $ $ $ F E e S E 2 Times New Roman Symbol & Arial & France p- " h