革兰氏阴性细菌质量控制因子SurA和FkpA等在外膜蛋白质生成过程中的功能及机制的在体研究

细菌外膜蛋白生成过程异常复杂，其质量控制极其重要。对β-桶外膜蛋白质生成的膜间质质量控制机制的认识还远不清楚,活体研究更是缺乏。本申请以大肠杆菌为模式生物，以膜间质分子伴侣SurA和FkpA为主要研究对象，在活细胞体内研究β-桶外膜蛋白质生成过程质量控制机制。具体内容包括：1、SurA和FkpA在活细胞内的天然相互作用蛋白质全谱鉴定；2、揭示SurA和FkpA参与β-桶外膜蛋白生成途径的分子机制；3、SurA和FkpA之间，及其与其它质量控制因子之间的协同作用机制；4、研究FkpA的功能重要性在热胁迫条件下凸显的机制。本研究中，我们将利用非天然氨基酸体内光交联技术。这是我们实验室多年来使用研究活细胞内蛋白质相互作用的有效方法。通过此项技术，我们可以实现在体内捕获和SurA、FkpA相互作用的蛋白质，包括天然底物和相互作用的功能因子，以此认识它们的功能和作用。

The biogenesis of the outer membrane proteins in Gram negative cells is considered a rather complex processes and thus their quality control is vital for opetimal cell growth. The quality control for the biogenesis of the beta-barrel outer membrane proteins is far from clear and in vivo study is especially lacking.This application will use E. coli as the model organism and focusing on two periplasmic quality control factors, SurA and FkpA. We will mainly conduct research on the following aspects: 1. to identify all the proteins interacting with them in living cells; 2. To understand their role in the biogenesis of beta-barrel outer membrane proteins; 3. to unveil the cooperation between SurA, FkpA and other quality control factors; 4. To characterize the unique role of FkpA under heat shock conditions。 We will perform our studies by employing the in vivo photo-crosslinking method using genetically incorporated non-natural amino acids,a method that we having been using in our lab to examine protein-protein interactions in living cells. This will allow us to identify all the proteins interacting with SurA and FkpA, thus to understand their function and action mechanism in living cells for the quality control of outer membrane proteins.