Dissecting the Structure and Function of the PDU Microcompartment in Salmonella

描述(申请人提供)：细菌微室是由代谢酶组成的大的亚细胞结构，包裹在由多个亚基组成的蛋白质外壳中。它们在细菌中广泛存在，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理。它们的功能是隔离和调节某些代谢途径中发现的有毒或挥发性中间体的产生。然而，对于这是如何在机制层面上发生的，我们知之甚少。这项拟议研究的长期目标是阐明沙门氏菌降解1，2-丙二醇的分子原理并建立微室的三维结构。沙门氏菌系统在可用于微格机械研究的知识和工具方面是无与伦比的。拟议的研究结合了遗传学、生物物理学和结构学方法，在机制水平上阐明了沙门氏菌PDU微隔室的细胞功能。提出了三个具体的目标：1.确定末端螺旋和其他将蛋白质靶向PDU微室腔的机制的作用；2.确定毛孔和辅因子循环在为PDU微室的管腔酶提供所需底物和辅助因子方面的作用；以及3.阐明PDU微室的高阶结构和组装。结构将通过X射线结晶学、生物物理学和计算方法进行研究和分析。蛋白质-蛋白质结合研究将包括His-tag下拉、生物物理方法和结晶学。我们将结合遗传和生化研究，从结构导向的突变中获得功能和机制方面的见解。完成拟议的调查将阐明沙门氏菌PDU微格的机理和结构原理。这将提供对细菌微隔室的一般见解。由于细菌微室在许多微生物中发挥着关键的代谢作用，包括几种人类病原体，拟议的研究可能最终会带来干扰致病过程的新机会。 与公共卫生相关：对细菌微室的更好理解，可能最终导致干扰致病过程的新机会，也可能为合理设计用于药物生产或作为药物输送载体的合成蛋白质笼提供基础

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: An improved understanding of bacterial microcompartments, which are found in many human pathogens, may ultimately lead to new opportunities for interfering with pathogenic processes, and may also provide a basis for the rational design of synthetic protein cages for use in the production of pharmaceuticals or as drug delivery vehicles