Dissecting the Structure and Function of the PDU Microcompartment in Salmonella

项目概述：细菌微区室是由以下组成的大型亚细胞结构： 由多个亚基组成的蛋白质壳内包裹的代谢酶。他们是 广泛存在于细菌中，功能多样，与发病机制有关，具有许多 重要的潜在生物医学应用，并似乎结合了独特的机制和 结构原则。它们的功能是隔离和调节有毒或挥发性物质的产生， 在某些代谢途径中发现的中间体。然而，很少有人知道这是如何发生的。 发生在机械层面。拟议研究的长期目标是阐明 分子原理，并建立微区室的三维结构 参与沙门氏菌降解1，2-丙二醇。沙门氏菌系统是无与伦比的 关于微区室机理研究的知识和工具。 拟议的研究结合联合收割机遗传，生物物理和结构的方法来阐明 沙门氏菌Pdu微区室在机制水平上的细胞功能。三个具体 提出了以下目标：1.确定末端螺旋和其他靶向机制的作用 蛋白质到Pdu微区室的内腔; 2.确定孔隙和辅因子的作用 再循环为Pdu微区室的管腔酶提供所需的 底物和辅因子;和3.阐明Pdu的高阶结构和组装 微区室将通过X射线晶体学研究和分析结构， 生物物理和计算方法。蛋白质-蛋白质结合研究将包括His-标签 下拉、生物物理学方法和晶体学。功能和机械的见解将 来自结构导向诱变结合遗传和生物化学 问题研究完成拟议的调查将阐明机械和结构 沙门氏菌pdu微区室的原理。这将提供一般的见解， 细菌微区室由于细菌微区室在代谢中起着关键的作用， 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了新的机会。

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.