Bacterial back talk - unfolding and molecular interaction studies in Staphylococcal proteins and engineering structure-informed antibiotics

细菌顶嘴——葡萄球菌蛋白和工程结构知情抗生素的展开和分子相互作用研究

• 批准号: 2182232
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2182232
• 关键词: Bacterial; back; talk; unfolding; molecular

Staphylococci are extremely common human commensals, which have been implicated in systemic infections in hospitals, biofilm formation in surgical devices and implants, industrial fouling and food spoilage. The accessory gene regulation (Agr) in staphylococci controls important bacterial functions, such as virulence and adhesion to surfaces (biofilm formation). AgrB is a membrane endopeptidase responsible for pre-processing of the signalling molecule, AIP, which mediates bacterial communications and quorum sensing. The AIP response cascade is a two component system including a membrane receptor AgrC, which phosphorylates the nucleotide-binding partner AgrA. Pharmacological regulation of the Agr protein is an attractive target for bacterial control, attenuating virulence and biofilm inhibition and a new opportunity for antimicrobial design.We have developed a new molecular recognition assay, which reports on substrate or ligand binding through increased protein stability to degradation. Thermal unfolding of proteins is a gradual process, in which thermal fluctuations increase in frequency and magnitude with temperature and can be monitored by circular dichroism, small angle neutron or X-ray scattering. NMR spectroscopy and high performance computing complement the studies with detailed atomic level structural information on the interaction between the Agr proteins and their substrates and regulatory ligands. In this project we will develop a large scale screen for protein/ligand interactions based on the thermal denaturation assay and will validate it against existing libraries of Agr modulators. Using recombinant protein from current research project on Agr, we will:a) use a combination of scattering techniques and CD to monitor changes in protein structure and overall conformation and will develop the tool with reference to critical behavior;b) develop a sample environment at partner institution ISIS for the large scale screen in conjunction with beamlines SANS2D and ZOOM; develop analytical framework for rapid data interpretation;c) characterize ligand/receptor interactions using solid state NMR, molecular docking and simulations.The programme relies on advanced biophysical, structural and computational tools for molecular analysis, which are combined in complementary, yet partially overlapping groups to ensure successful completion of the work.

葡萄球菌是极其常见的人类共生菌，与医院的系统性感染、手术器械和植入物中生物膜的形成、工业污染和食品腐败有关。葡萄球菌中的辅助基因调控 (Agr) 控制重要的细菌功能，例如毒力和表面粘附（生物膜形成）。 AgrB 是一种膜内肽酶，负责信号分子 AIP 的预处理，介导细菌通讯和群体感应。 AIP 反应级联是一个两部分系统，包括膜受体 AgrC，其磷酸化核苷酸结合伴侣 AgrA。 Agr 蛋白的药理学调节是细菌控制、减弱毒力和生物膜抑制的一个有吸引力的目标，也是抗菌设计的新机会。我们开发了一种新的分子识别测定法，它通过增加蛋白质降解稳定性来报告底物或配体结合。蛋白质的热解折叠是一个渐进的过程，其中热波动的频率和幅度随着温度的增加而增加，可以通过圆二色性、小角中子或 X 射线散射来监测。 NMR 光谱和高性能计算通过有关 Agr 蛋白及其底物和调节配体之间相互作用的详细原子级结构信息补充了研究。在这个项目中，我们将开发基于热变性测定的蛋白质/配体相互作用的大规模筛选，并将根据现有的 Agr 调节剂库对其进行验证。使用当前 Agr 研究项目中的重组蛋白，我们将：a) 结合使用散射技术和 CD 来监测蛋白质结构和整体构象的变化，并将开发参考关键行为的工具；b) 开发一个样本环境合作伙伴机构 ISIS，用于与光束线 SANS2D 和 ZOOM 结合的大型屏幕；开发快速数据解释的分析框架；c) 使用固态核磁共振、分子对接和模拟来表征配体/受体相互作用。该程序依赖于先进的生物物理、结构和计算工具进行分子分析，这些工具组合成互补但部分重叠的组以确保工作的顺利完成。