Dissecting the Structure and Function of the PDU Microcompartment in Salmonella

解析沙门氏菌 PDU 微区室的结构和功能

• 批准号: 8839176
• 负责人: THOMAS Aquinas BOBIK
• 金额: $ 48.45万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839176
• 关键词: Amino Acids; Bacteria; Binding; Biochemical; Caliber; Cell physiology; Computing Methodologies; Crystallography; Diffusion; Drug Delivery Systems; Encapsulated; Enzymes; Family; Genes; Genetic; Goals; Higher Order Chromatin Structure; Homeostasis; Homologous Gene; Human; In Vitro; Investigation; Knowledge; Lead; Link; Mediating; Metabolic; Metabolic Pathway; Methods; Microbe; Modeling; Molecular; Movement; Mutagenesis; N-terminal; NADH; Organelles; Pathogenesis; Pharmacologic Substance; Play; Process; Production; Propylene Glycols; Protein Binding; Protein-Protein Interaction Map; Proteins; Publishing; Recycling; Research; Role; Salmonella; Salmonella enterica; Salmonella typhimurium LT2; Site-Directed Mutagenesis; Specificity; Structure; Subcellular structure; System; Testing; Work; base; biophysical techniques; cofactor; cytotoxicity; design; enzyme substrate; improved; in vivo; insight; mutant; pathogen; prevent; structural biology; synthetic protein; targeted sequencing; three dimensional structure; tool

Project Summary: Bacterial microcompartments are large subcellular structures composed of metabolic enzymes encapsulated within a protein shell built from multiple subunits. They are widespread among bacteria, functionally diverse, linked to pathogenesis, have a number of important potential biomedical applications, and appear to incorporate unique mechanistic and structural principles. Their function is to sequester and regulate the production of toxic or volatile intermediates found in certain metabolic pathways. However, little is known about how this is occurs at the mechanistic level. The long-term goal of the proposed research is to elucidate the molecular principles and to build up a 3-dimensional structure of the microcompartments involved in 1,2-propanediol degradation by Salmonella. The Salmonella system is unmatched with regard to the knowledge and tools available for mechanistic studies of microcompartments. The proposed studies combine genetic, biophysical, and structural methods to elucidate the cellular function of the Salmonella Pdu microcompartment at a mechanistic level. Three specific aims are proposed: 1. Determine the role of terminal helixes and other mechanisms for targeting proteins to the lumen of the Pdu microcompartment; 2. Determine the role of pores and cofactor recycling in supplying the lumen enzymes of the Pdu microcompartment with required substrates and cofactors; and 3. Elucidate the higher order structure and assembly of the Pdu microcompartment. Structures will be investigated and analyzed by x-ray crystallography, biophysical, and computational methods. Protein-protein binding studies will include his-tag pulldowns, biophysical methods, and crystallography. Functional and mechanistic insights will be derived from structure-guided mutagenesis in conjunction with genetic and biochemical studies. Completion of the proposed investigations will elucidate the mechanistic and structural principles of the Salmonella pdu microcompartment. This will provide general insights into bacterial microcompartments. Since bacterial microcompartments play critical metabolic roles in many microbes, including several human pathogens, the proposed studies may ultimately lead to new opportunities for interfering with pathogenic processes.

项目摘要：细菌微型室是由大型亚细胞结构组成的 代谢酶封装在由多个亚基建造的蛋白质壳中。他们是 在细菌中广泛存在，功能多样，与发病机理相关，具有许多 重要的潜在生物医学应用，并且似乎包含了独特的机械和 结构原理。它们的功能是隔离和调节有毒或挥发性的产生 在某些代谢途径中发现的中间体。但是，对这是什么知之甚少 发生在机械水平。拟议研究的长期目标是阐明 分子原理并建立微校区的3维结构 沙门氏菌参与1,2-丙二醇降解。沙门氏菌系统无与伦比 关于可用于微型企业的机械研究的知识和工具。 提出的研究结合了遗传，生物物理和结构方法，以阐明 沙门氏菌PDU微校区在机械水平上的细胞功能。三个具体 提出了目的：1。确定终端螺旋的作用和其他机制的靶向 蛋白质到PDU微型室的腔内； 2。确定孔和辅因子的作用 回收PDU微校区的腔内酶 底物和辅因子；和3。阐明PDU的高阶结构和组装 微型室。结构将通过X射线晶体学研究和分析， 生物物理和计算方法。蛋白质 - 蛋白质结合研究将包括His-TAG 下拉，生物物理方法和晶体学。功能和机械洞察力将 源自结构引导的诱变与遗传和生化 研究。拟议的调查的完成将阐明机械和结构 沙门氏菌PDU微室的原理。这将提供一般的见解 细菌微型室。由于细菌微型室在 许多微生物，包括几种人类病原体，提出的研究可能最终领导 为干扰致病过程的新机会。