Dissecting the Structure and Function of the PDU Microcompartment in Salmonella

项目摘要：细菌微室是一种大型亚细胞结构，由 代谢酶被包裹在由多个亚基组成的蛋白质外壳中。他们是 在细菌中广泛存在，功能多样，与发病机制有关，有许多 重要的潜在生物医学应用，并似乎结合了独特的机械和 结构性原则。它们的功能是隔离和调节有毒或挥发性物质的生产 在某些代谢途径中发现的中间产物。然而，人们对这是如何发生的知之甚少。 发生在机械学层面上。拟议研究的长期目标是阐明 分子原理和建立微室的三维结构 参与沙门氏菌降解1，2-丙二醇。沙门氏菌体系无与伦比 关于可用于微格机械研究的知识和工具。 拟议的研究结合了遗传、生物物理和结构方法来阐明 沙门氏菌PDU微室在机械水平上的细胞功能。三个具体的 目标是：1.确定末端螺旋和其他靶向机制的作用 蛋白质进入PDU微腔；2.确定毛孔和辅因子的作用 回收供应PDU微室所需的管腔酶 底物和辅因子；3.阐明PDU的高阶结构和组装 微型隔间。结构将通过X射线结晶学进行研究和分析， 生物物理学和计算方法。蛋白质-蛋白质结合研究将包括组氨酸标签 下拉菜单、生物物理方法和结晶学。功能和机械的洞察力将 是由结构导向突变与遗传和生化结合而来的 学习。拟议的调查工作完成后，将会阐明 沙门氏菌PDU微隔室的原理。这将提供对以下方面的一般见解 细菌微格。由于细菌微室在体内起着关键的代谢作用 许多微生物，包括几种人类病原体，拟议的研究可能最终导致 为干扰致病过程提供新的机会。

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.