Computational Assembly of Beta Barrel Membrane Protein

描述(申请人提供)：在革兰氏阴性细菌、抗酸革兰氏阳性细菌、真核细胞线粒体、叶绿体(例如，在大肠杆菌、脑膜炎奈瑟氏菌、淋病奈瑟氏菌和结核分枝杆菌等分枝杆菌中)的外膜中发现桶。革兰氏阳性菌的许多致孔外毒素也是膜蛋白(如金黄色葡萄球菌的溶血素和炭疽杆菌的保护性抗原)。桶状膜蛋白在许多基本的生物过程中都是重要的。它们控制着离子和有机分子在细菌和线粒体外膜上的交换和运输。它们对除古生菌以外的所有生命领域的蛋白质转运都是必不可少的。它们调节新陈代谢和细胞凋亡。它们对免疫监测、对抗生素的抗药性也很重要，也是细菌毒力的关键决定因素。因此，β-桶膜蛋白是开发抗传染病药物和疫苗的重要治疗靶点。在开发用于高通量DNA测序的生物纳米孔以及用于靶向抗癌药物输送的纳米设备方面，它们也是重大工程努力的重点。尽管已经通过实验和计算研究了解到了很多东西，但目前关于β-桶膜蛋白的知识还不完整，只有一小部分结构已知，而且对β-桶膜蛋白的一般组织原理缺乏了解。这项研究的长期目标是对β-桶膜蛋白的结构、相互作用和功能有基础的了解和机制上的洞察，并开发设计具有增强生物物理性质的β-桶膜蛋白的技术。其具体目标是：1)开发控制？-桶膜蛋白组装的物理原理的计算模型。粗粒模型将被开发来考虑结构稳定性和蛋白质-蛋白质相互作用(PPI)的关键决定因素。这将使通过计算对蛋白质稳定性进行定量评估成为可能。2)预测β-桶状膜蛋白的结构、齐聚状态和蛋白-蛋白质界面。重点将放在预测新型建筑结构的挑战性任务上 或没有已知模板的结构。此外，还将开发预测蛋白质齐聚状态和确定蛋白质-蛋白质相互作用位置的方法。具有已知模板的结构也将通过使用新开发的进化分析技术检测远程同源基因来预测。3)发展设计具有良好稳定性、齐聚状态和孔几何形状的桶状孔的工程原理。将开发具有不同齐聚状态和不同稳定性的？桶膜孔蛋白的设计策略。将设计出稳定性增强和稳定性减弱的蛋白质，包括单体蛋白质和寡聚蛋白质。此外，还将设计具有复杂孔道几何结构的孔蛋白，这些孔道几何结构使用自然生成的构建块。4)计算预测和设计的实验验证。计算预测将通过实验研究得到验证。将进行广泛的突变研究，以测试设计的桶状膜蛋白在稳定性、寡聚化状态和几何构型方面是否具有预期的变化。

DESCRIPTION (provided by applicant): Barrels are found in the outer membrane of gram-negative bacteria, acid-fast gram-positive bacteria, eukaryotic mitochondria, chloroplasts (e.g., in E. coli, N. meningitidis, N. gonorrheae, and mycobacteria such as M. tuberculosis). Many pore-forming exotoxins from gram-positive bacteria are also -barrel membrane proteins (e.g., ?-hemolysin of S. aureus and protective antigen of B. anthracis). ?-barrel membrane proteins are important for many fundamental biological processes. They control the ex- change and transport of ions and organic molecules across the bacterial and mitochondrial outer membranes. They are essential for protein translocation in all domains of life, except archaea. They regulate metabolism and apoptosis. They are also important for immune surveillance, in providing resistance to antibiotics, and are key determinants of bacterial virulence. As a result, ?-barrel membrane proteins are important therapeutic targets for developing drugs and vaccines against infectious diseases. They are also the focus of significant engineering efforts in developing biological nanopores for high-throughput DNA sequencing, as well as nano-devices for targeted cancer drug delivery. Although much has been learned through experimental and computational studies, current knowledge of ?-barrel membrane proteins is incomplete Only a small number of structures are known, and there is a lack of understanding of the general organizing principles of ?-barrel membrane proteins. The long term goal of the proposed research is to gain fundamental understanding and mechanistic insight into the structures, interactions, and functions of ?-barrel membrane proteins, and to develop enabling technology for design of ?-barrel membrane proteins with enhanced biophysical properties. The specific aims are to: 1) Develop computational models of physical principles governing the assembly of ?-barrel membrane proteins. Coarse-grained models will be developed to account for key determinants of structural stability and protein-protein interactions (PPIs) of ?-barrel membrane proteins. This will enable quantitative assessment of protein stability through computation. 2) Predict structures, oligomerization state, and protein-protein interfaces of ?-barrel membrane proteins. The focus will be on the challenging tasks of predicting structures of novel architecture or structures with no known templates. In addition, methods will be developed to predict protein oligomerization states and to identify protein-protein interaction sites. Structures with known templates will also be predicted through detection of remote homologs using newly developed technique of evolutionary analysis. 3) Develop engineering principles for designing ?-barrel porins with desirable stability, oligomerization state, and pore geometry. Design strategies for ?-barrel membrane porins with altered oligomerization states and altered stability will be developed. Proteins with enhanced as well as weakened stability, for both monomeric and oligomeric proteins will be designed. In addition, porins with complex pore geometry using naturally occurring building blocks will also be designed. 4) Experimental validation of computational prediction and design. Computational predictions will be verified by experimental studies. Extensive mutant studies will be carried out to test whether designed ?-barrel membrane proteins have the intended changes in stability, in oligomerization state, as well as in geometry.