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: 6138519
• 负责人: KOTTAYIL I VARUGHESE
• 金额: $ 23.66万
• 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 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138519
• 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：这项提议的目标是使用X光 用结晶学技术研究细胞的结构和功能 枯草芽孢杆菌孢子形成过程中的调节蛋白。这个 产孢子的调节开关的主要组件是ATP 依赖的激酶和KinB，以及反应调节蛋白- Spo0F、Spo0B和Spo0A。当肌动蛋白被激活时，磷光传递被启动 使第二信使Spo0F磷酸化。Spo0F-P是 Spo0B，一种磷酸蛋白磷酸转移酶，将其磷酸盐转移到 Spo0A中的关键天冬氨酸残基。当增长条件恶化时， 细胞内Spo0A-P水平升高，ABB基因受到抑制， 产孢子基因的转录被启动。Spo0A是唯一的 转录因子在这个复杂的细胞孢子形成过程中。 该提案将重点介绍Spo0F、Spo0B和Spo0B的结构和性能 Spo0A。需要解决的关键问题是(I)是什么使磷酸盐 在Spo0F中比在趋化类似蛋白中更稳定，Chey？ (Ii)磷向Spo0F转移和从Spo0F转移的机制是什么？立地 定向突变将被用来确定在生物体中重要的残基 与激酶、Spo0B和磷酸酶的相互作用，(Iii)他的残基得到的 在Spo0B中被磷酸化，这种残基的环境是什么？(四) Spo0A N-末端结构域的磷酸化如何影响 其C-末端结构域的DNA结合特性？ 测定了Spo0F的晶体结构。此外， 一个Y13S耐药突变株的结构研究 Spo0L也已确定。Spo0B已经结晶。绕射 天然晶体数据为2.9埃，黄金衍生品为3.0埃 埃斯特罗姆，铂的导数为3.2埃，汞的导数为3.8埃 已经被测量过了。 Epo0A是一个具有两个结构域的蛋白质。N-末端结构域与Spo0F同源 还含有一种可被磷酸化的天冬氨酸。这个 C末端结构域包含与DNA相互作用的部分。N和 Spo0A的C末端结构域和整个蛋白在 E.Coli，并进行纯化。作为第一步，结晶正在进行中 向结构特征转变。 E