Stabilization and regulation of a Bacillus anthracis spore lytic enzyme

消除炭疽杆菌孢子和其他致病细菌， 由于孢子的极端抵抗力， 常规的抗菌处理。激活B。炭疽孢子皮层肽聚糖 裂解机制导致孢子休眠和抗性特性的快速丧失。这 因此，机器可以作为萌发激活和孢子杀灭处理的目标。的 皮层溶解酶SleB以无活性和高度稳定的形式储存在休眠孢子中， 在发芽过程中很早就变得活跃。YpeB蛋白是必需的， 将SleB掺入休眠孢子中，并且在萌发期间， YpeB释放SleB以使皮质去角质。拟议工作的目标1包括 生物化学、遗传学和结构生物学方法来揭示形成的蛋白质-蛋白质接触 休眠孢子中的YpeB和SleB。含有YpeB或SleB的蛋白质复合物将是 将鉴定从孢子和共纯化的蛋白质中纯化的。YpeB的改变形式将是 在体内表达，以确定参与功能和蛋白质的关键区域， 互动将使用X射线晶体学确定YpeB结构，以便 了解突变的影响。这项工作将阐明SleB稳定的机制， 其可适用于生产稳定的蛋白质药物、疫苗， 其他工业产品。目的2是鉴定加工YpeB的蛋白酶， 从而激活SleB。对这种蛋白酶及其调节的了解将为蛋白酶的制备提供方法。 通过YpeB和SleB激活萌发。拟议的工作将有助于两个主要的 翻译目标：激活孢子萌发，以改善去污和 了解蛋白质极端稳定的机制。

Elimination of spores of Bacillus anthracis and other disease-causing bacteria from contaminated sites or materials is challenging due to the extreme resistance of spores to conventional antimicrobial treatments. Activation of the B. anthracis spore cortex peptidoglycan lytic machinery results in rapid loss of spore dormancy and resistance properties. This machinery can thus be targeted for germination-activating and spore-killing treatments. The cortex lytic enzyme SleB is stored in an inactive and highly stable form in the dormant spore and becomes active early during the germination process. The YpeB protein is required for incorporation of SleB into the dormant spore, and during germination, proteolytic cleavage of YpeB releases SleB to depolymerize the cortex. Aim 1 of the proposed work includes biochemical, genetic, and structural biology methods to reveal protein-protein contacts formed by YpeB and SleB in the dormant spore. Protein complexes containing YpeB or SleB will be purified from spores and co-purified proteins will be identified. Altered forms of YpeB will be expressed in vivo to identify key regions of the protein involved in function and protein interaction. The YpeB structure will be determined using X-ray crystallography in order to understand the effects of mutations. This work will clarify the mechanism of SleB stabilization, which may be adaptable to the production of stable protein pharmaceuticals, vaccines, and other industrial products. Aim 2 is the identification of the protease that processes YpeB and thus activates SleB. Knowledge of this protease and its regulation will suggest methods for the activation of germination via YpeB and SleB. The proposed work will contribute to two major translational goals: Activation of spore germination in order to improve decontamination and understanding of a mechanism of extreme protein stabilization.