METALLOPROTEIN BASED INTERFACIAL SYSTEMS

基于金属蛋白的界面系统

• 批准号: 6478961
• 负责人: PATRICIA A MABROUK
• 金额: $ 5.36万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478961
• 关键词: bacteria; bioengineering /biomedical engineering; biological products; biomaterials; biomedical resource; biotechnology; carbohydrates; infection; proteins; structural biology; technology /technique

The long term objectives of the project are characterization of the enzymatic activities and gene products involved in synthesis of the peptidoglycan cell wall of bacterial endospores and examination of the role of each type of peptidoglycan structural modification in determining endospore resistance properties. The specific aims are 1) to determine the structure of the endospore peptidoglycan in the first stages of its synthesis and to track the structure to its final form in the dormant spore, 2) to identify changes in synthesis of this structure produced by mutation or loss of each penicillin-binding protein, autolysin, and sporulation-associated gene product, and 3) to purify and characterize in vitro the enzymatic activities involved in peptidoglycan side chain cleavage and muramic lactarn production. Knowledge of principles of endospore resistance properties, dormancy, and longevity may contribute to better decontamination methods and to methods for storage and transport of drugs and vaccines. Peptidoglycan synthesis in general is an attractive target for antibiotic action, further studies of this process will contribute to methods for identification of new antibiotics. Peptidoglycan is purified from sporulating cultures of Bacillus subtilis by chemical and enzymatic treatments, digested with muramidase, and analyzed using high-pressure liquid chromatography (HPLC) using methods previously developed for analysis of dormant spore peptidoglycan. Novel muropeptides are identified using amino acid analysis and mass spectrometry. Appearance and loss of peptide side chain alanine and glycine residues, muramic lactarn production, and changes in peptide cross-linking are quantified throughout the sporulation process. The analysis will be repeated using strains with altered or absent penicillin-binding proteins, autolysins, and sporulation-associated gene products. The cwlD and other gene products found to be associated with muramic lactam production will be assayed in vitro using extracts of sporulating cells or following over-expression in B. subtilis or Escherichia coli. Immature peptidoglycan samples from mutant strains and purified muropeptides will serve as substrates in these assays. Reaction progress will be monitored using the HPLC method and by development of a more rapid capillary electrophoresis method.

该项目的长期目标是 参与合成的酶活性和基因产物 细菌内生孢子的肽聚糖细胞壁及其检查 每种类型的肽聚糖结构修饰的作用 确定内生孢子抗性特性。 具体目标是1） 首先确定内生孢子肽聚糖的结构 其合成阶段并跟踪结构直至其最终形式 在休眠孢子中，2) 识别该孢子合成的变化 由每个青霉素结合突变或缺失产生的结构 蛋白质、自溶素和孢子形成相关基因产物，以及3) 体外纯化和表征涉及的酶活性 肽聚糖侧链裂解和胞壁内酰胺的产生。 了解内生孢子抵抗特性、休眠、 和长寿可能有助于更好的去污方法和 药物和疫苗的储存和运输方法。 一般来说，肽聚糖合成是一个有吸引力的目标 抗生素作用，对该过程的进一步研究将有助于 新型抗生素的鉴定方法. 肽聚糖是 通过化学方法从枯草芽孢杆菌的孢子培养物中纯化 和酶处理，用胞壁酶消化，并使用 使用以前的方法进行高压液相色谱 (HPLC) 开发用于分析休眠孢子肽聚糖。 小说 使用氨基酸分析和质量来鉴定胞肽 光谱测定法。 肽侧链丙氨酸的出现和丢失 甘氨酸残基、胞壁内酰胺的产生和肽的变化 交联在整个孢子形成过程中被量化。 这 将使用改变或缺失的菌株重复分析 青霉素结合蛋白、自溶素和孢子形成相关蛋白 基因产物。 cwlD 和其他基因产物被发现 与胞壁内酰胺产生相关的物质将在体外进行测定 使用孢子细胞提取物或在 B. 枯草芽孢杆菌或大肠杆菌。 未成熟肽聚糖样品来自 突变菌株和纯化的胞肽将作为底物 这些测定。 将使用 HPLC 监测反应进度 方法并通过开发更快速的毛细管电泳 方法。