STRUCTURE OF SPORULATION PROTEINS IN B SUBTILIS

枯草芽孢杆菌中孢子形成蛋白的结构

• 批准号: 2634806
• 负责人: KOTTAYIL I VARUGHESE
• 金额: $ 22.32万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634806
• 关键词: Bacillus subtilis; X ray crystallography; active sites; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; gene mutation; phosphoproteins; protein structure function; sporogenesis; structural biology; transcription factor

DESCRIPTION: The objective of this proposal is to use X-ray crystallographic techniques to study the structure and function of the regulatory proteins involved in the sporulation of Bacillus subtilis. The main components of the regulatory switch for sporulation are the ATP dependent kinases KinA and KinB, and the response regulatory proteins - Spo0F, Spo0B and Spo0A. The phosphorelay is initiated when the kinases phosphorylate the second messenger Spo0F. Spo0F-P is the substrate for Spo0B, a phosphoprotein phosphotransferase which transfers its phosphate to a key Asp residue in Spo0A. When conditions for growth deteriorate, the cellular levels of Spo0A-P increase and the abB gene becomes repressed and transcription of the sporulation genes are initiated. Spo0A is the sole transcription factor in this complex cellular process of sporulation. The proposal will focus on the structure and properties of Spo0F, Spo0B and Spo0A. The key questions to be resolved are (i) What makes the phosphate more stable in Spo0F than in the analogous protein of chemotaxis, CheY? (ii) What is the mechanism of phosphate transfer to and from Spo0F? Site directed mutations will be employed to identify residues important in the interactions with KinA, Spo0B and phophatases, (iii) Which His residue gets phosphorylated in Spo0B and what is the environment of this residue? (iv) How does the phosphorylation of the N-terminal domain of Spo0A influence the DNA binding properties of its C-terminal domain? The crystal structure of Spo0F has been determined. In addition, the structure of a Y13S mutant that is resistant to the phosphatase activity of Spo0L has also been determined. Spo0B has been crystallized. Diffraction data on the native crystal to 2.9 Angstrom, a gold derivative to 3.0 Angstrom, a Pt derivative to 3.2 Angstrom and Hg derivative to 3.8 Angstrom have been measured already. Epo0A is a two domain protein. The N-terminal domain is homologous to Spo0F and also contains an aspartic acid that can be phosphorylated. The C-terminal domain contains the DNA interacting portions. The N and C-terminal domains of Spo0A and the whole protein have been overexpressed in E. coli and purified. Crystallization is in progress as the first step towards structural characterization. E

描述：本提案的目的是使用X射线 晶体学技术来研究的结构和功能， 参与枯草芽孢杆菌孢子形成的调节蛋白。 的 孢子形成调节开关的主要成分是ATP 依赖性激酶KinA和KinB，以及反应调节蛋白- Spo0F、Spo0B和Spo0A。 磷酸化继电器启动时，激酶 磷酸化第二信使Spo0F。 Spo0F-P是用于 Spo0B，一种磷蛋白磷酸转移酶，将其磷酸转移到 Spo0A中关键的Asp残基。 当增长条件恶化时， Spo0A-P的细胞水平增加，abB基因被抑制， 孢子形成基因的转录开始。 Spo0A是唯一的 转录因子在这个复杂的细胞孢子形成过程中的作用。 该提案将集中在Spo0F，Spo0B和Spo0C的结构和性质上。 Spo0A。 需要解决的关键问题是（i）是什么使磷酸盐 更稳定的Spo0F比类似的蛋白质的趋化性，CheY？ (ii)磷酸盐转移到Spo0F和从Spo0F转移的机制是什么？ 网站 定向突变将用于鉴定在所述细胞中重要的残基。 与KinA，Spo0B和磷酸酶的相互作用，（iii）His残基得到 Spo0B中的磷酸化，这个残基的环境是什么？ （四） Spo0A的N端结构域的磷酸化如何影响Spo0A蛋白的表达？ 其C-末端结构域的DNA结合特性？ 测定了Spo0F的晶体结构。 此外该 Y13S突变体的结构，该突变体对以下磷酸酶活性具有抗性： Spo0L也已确定。 Spo0B已经结晶。 衍射 天然晶体上的数据为2.9埃，金衍生物为3.0埃 埃，铂衍生物为3.2埃，汞衍生物为3.8埃 已经被测量过了。 Epo0A是一个双结构域蛋白。 N-末端结构域与Spo0F同源 并且还含有可被磷酸化的天冬氨酸。 的 C-末端结构域包含DNA相互作用部分。 n和 Spo0A和整个蛋白质的C-末端结构域已经在大肠杆菌中过表达。 e. coli中表达并纯化。 第一步是结晶 结构特征。 e