The Role of N-terminal Phosphorylation of the Photosystem IIProteins CP-43 and D2 as Probed by Site-Directed Mutagenesis

9407187普特南-埃文斯这是一项研究计划拨款。该项目的重点是在光系统II蛋白CP43和D2中缺乏n端苏氨酸残基的聚胞菌突变体的生产和初步表征。在高等植物中，这些苏氨酸残基在CP43和D2中被磷酸化。目前尚不清楚这种磷酸化在PSII功能中可能起什么作用，尽管已经提出了一些假设。该项目的目的是确定这两种蛋白的可逆磷酸化/去磷酸化是否对聚囊藻的PS II功能很重要。这种蓝藻是一个明确的遗传模式系统，其光系统与高等植物的光系统具有很强的同源性。用各种方法改变的内源性基因代替内源性基因很容易完成，并为研究单个氨基酸或蛋白质内氨基酸的小片段的功能作用提供了一种手段。这些蛋白的CP-43和D2的n端苏氨酸残基在高等植物和蓝藻之间是保守的。寡核苷酸定向诱变将在体外用于在编码这些蛋白质的基因的n端苏氨酸密码子中产生突变。突变将与密切相关的抗生素耐药性标记一起重新引入聚胞菌。突变体将筛选抗生素耐药性和可能的光自养生长损失。PSII功能改变的突变体将被选择并进行更深入的研究。磷酸化，或在蛋白质的某些丝氨酸或苏氨酸残基上添加磷酸基团，是翻译后的事件。一般来说，磷酸化-去磷酸化反应是细胞控制某些酶或结构蛋白活性的手段。确定磷酸化对特定蛋白质功能的作用的一种方法是通过分子遗传操作创造缺乏适当位点（即丝氨酸或苏氨酸残基）的蛋白质变体，并寻找功能的改变。这种方法将用于研究光系统II的两种蛋白质磷酸化的作用，光系统II是叶绿体中的多蛋白质复合物，负责为地球上大多数生命提供化学能的光合反应。如果成功，研究结果将提供关于细胞如何调节光合作用过程的重要信息。＊＊＊

9407187 Putnam-Evans This is a Research Planning Grant. The project focuses on the production and preliminary characterization of Synechocystis mutants lacking the N-terminal threonine residues in the Photosystem II proteins CP43 and D2. These threonine residues are phosphorylated in CP43 and D2 in higher plants. It is not yet clear what role(s) this phosphorylation may play in PSII function, although a number of hypotheses have been put forward. The goal of the project is to determine whether reversible phosphorylation/dephosphorylation of these two proteins is important to PS II function in Synechocystis. This cyanobacterium is a well defined genetic model system whose photosystems bear strong homology to those of higher plants. Replacement of endogenous genes with ones that have been altered by various methods is easily accomplished and provides a means for studying the functional role(s) of individual amino acids or small stretches of amino acids within proteins. The N-terminal threonine residue in both CP-43 and D2 of these proteins is conserved between higher plants and cyanobacteria. Oligonucleotide-directed mutagenesis will be employed in vitro to produce mutations in the N-terminal threonine codon in the genes encoding these proteins. Mutations will be re-introduced into Synechocystis along with a closely linked antibiotic resistance marker. Mutants will be screened for antibiotic resistance and possible loss of photoautotrophic growth. Mutants with altered PSII function will be selected and studied in greater depth. %%% Phosphorylation, or the addition of phosphate groups to certain serine or threonine residues of proteins, is a post- translational event. Phosphorylation-dephosphorylation reactions, in general, are means whereby cells control the activity of certain enzymes or structural proteins. One way to determine the role of phosphorylation on the function of a specific protein is to create a variant of the protein which lacks th e appropriate site (i.e., the serine or threonine residue), by molecular genetic manipulation, and look for alterations in function. This approach will be used to look at the role of phosphorylation in two proteins of Photosystem II, a multi-protein complex in chloroplasts which is responsible for the photosynthetic reactions that provide the chemical energy for most of life on earth. If successful, the results will be provide important information about how cells regulate the photosynthetic process. ***