A genomic and structural study of FtsZ constriction in cell division

细胞分裂中 FtsZ 收缩的基因组和结构研究

• 批准号: 8067948
• 负责人: Kiani Anela Jeniah Arkus Gardner
• 金额: $ 3.08万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8067948
• 关键词: Antibiotics; Bacteria; Bacterial Proteins; Base Pairing; Binding Sites; Bioinformatics; C-terminal; Caulobacter; Cell division; Cell membrane; Cells; Chromosome Mapping; Cleaved cell; Complement; Coupled; DNA Resequencing; Data; Dideoxy Chain Termination DNA Sequencing; Engineering; Escherichia coli; Fluorescence Microscopy; Frequencies; Generations; Genes; Genome; Genomics; Goals; Homologous Gene; In Vitro; Lateral; Length; Link; Liposomes; Maps; Membrane; Modeling; Mutation; Parents; Pathway interactions; Phenotype; Point Mutation; Prokaryotic Cells; Proteins; Rhizobium; Role; Site; Structure; Suppressor Mutations; System; TEV protease; Tail; Technology; Temperature; Testing; Thick; Tubulin; Variant; X-Ray Crystallography; adducin; constriction; flexibility; in vivo; insight; interest; large scale production; loss of function; loss of function mutation; mutant; novel; plasma protein Z; programs; public health relevance; reconstitution; structural genomics

DESCRIPTION (provided by applicant): FtsZ (filamentous temperature sensitive Z) is an essential protein for bacterial cell division. It assembles the Z-ring that pinches the cell in two. I am interested in two different aspects of FtsZ function. 1. FtsZ is the only protein necessary for constriction force generation in liposomes in vitro. FtsZ from foreign bacteria and some mutant FtsZ were also able to cause cell division in E. coli. However, some forms of FtsZ tested required the acquisition of unknown genomic mutations to suppress the defective FtsZ phenotype and allow division. The mutations in these suppressor strains are currently unknown, but are likely loss-of- function mutations in other pathways involved in cell division. This project aims to utilize high-throughput sequencing technology to resequence the entire genome of each of the 13 suppressor strains to map the suppressor mutation and identify further genes of interest in bacterial cell division. 2. The FtsZ protein has a C-terminal (Ct) tail region that lacks a lacks a well-defined structure as visualized through X-ray crystallography, indicating that it serves primarily as a spacer to link the FtsZ globular domain to the FtsA binding site and the membrane. However, some FtsZ mutants with small 20aa insertions in the Ct tail are incapable of functioning for cell division. This project aims to examine the role of the Ct tail function in vivo and in vitro. The Ct tail will be expressed and purified apart from the FtsZ globular domain for NMR structure determination. Varied forms of FtsZ will be expressed using alternative Ct tail sequences and length, and the ability of the protein to produce a constriction force in liposomes in vitro, and to function for cell division in vivo will be assessed. Public Health Relevance: FtsZ is a required protein for cell division in most bacteria. A detailed understanding of FtsZ structure and function would increase the possibility and efficacy of targeting the protein pharmacologically to inhibit bacterial division, thus creating potential new antibiotics.

描述（由申请人提供）：FtsZ（丝状温度敏感Z）是细菌细胞分裂的必需蛋白质。它组装将电池一分为二的 Z 形环。我对 FtsZ 功能的两个不同方面感兴趣。 1. FtsZ 是体外脂质体产生收缩力所必需的唯一蛋白质。来自外来细菌的 FtsZ 和一些突变体 FtsZ 也能够引起大肠杆菌中的细胞分裂。然而，某些形式的 FtsZ 测试需要获得未知的基因组突变来抑制有缺陷的 FtsZ 表型并允许分裂。这些抑制菌株中的突变目前未知，但可能是参与细胞分裂的其他途径的功能丧失突变。该项目旨在利用高通量测序技术对 13 种抑制菌株的整个基因组进行重新测序，以绘制抑制突变图谱并进一步鉴定细菌细胞分裂中感兴趣的基因。 2. FtsZ 蛋白有一个 C 末端 (Ct) 尾区，通过 X 射线晶体学观察，该区域缺乏明确的结构，表明它主要作为间隔区将 FtsZ 球状结构域连接到 FtsA 结合位点和膜。然而，一些在 Ct 尾部插入 20 个氨基酸的 FtsZ 突变体无法发挥细胞分裂的功能。该项目旨在研究 Ct 尾功能在体内和体外的作用。 Ct 尾部将与 FtsZ 球状结构域分开表达和纯化，用于 NMR 结构测定。将使用替代的 Ct 尾序列和长度来表达各种形式的 FtsZ，并且将评估该蛋白质在体外在脂质体中产生收缩力以及在体内发挥细胞分裂功能的能力。 公共健康相关性：FtsZ 是大多数细菌细胞分裂所需的蛋白质。对 FtsZ 结构和功能的详细了解将增加从药理学上靶向该蛋白质来抑制细菌分裂的可能性和功效，从而创造潜在的新型抗生素。