论文部分内容阅读
pH值是几乎影响到所有蛋白质分子表面电荷分布和相关结构变化的关键因素,许多蛋白质分子之间的相互作用也受到pH值的调控.近年来,基于非天然氨基酸的光交联探针被广泛应用于捕捉活细胞内的蛋白-蛋白相互作用.然而,由于环境pH值的改变往往导致蛋白质分子结构、带电性质的显著变化,因此现有的非天然氨基酸光交联探针难以实现在极端pH值条件下对相互作用的蛋白质分子的捕获和研究.本文将介绍本课题组新近发展的基于烷基双吖丙啶活性基团的非天然氨基酸光交联探针-DIZPK,通过这一探针,我们成功捕获到大肠杆菌中一种重要的酸性分子伴侣HdeA在膜间质内酸性胁迫过程中的作用对象.在捕获到的HdeA底物中,我们发现了两个膜间质中重要的分子伴侣蛋白:DegP和SurA.通过实验我们证明了在酸性胁迫条件下,DegP和SurA能够被HdeA保护不形成聚集体,并进而在随后的回复中性过程中能够协助HdeA对其他底物进行重折叠.这种不依赖于ATP的分子伴侣间协作模式可能起到了帮助肠道型细菌抵抗酸性胁迫的功能.基于上述实验结果,我们提出了一个“分子伴侣协同作用”的模型,用以阐释细菌利用抗酸性分子伴侣提高其在酸胁迫下逃逸的机理.推而广之,在原核和真核细胞中定点引入高可适性的非天然氨基酸光交联探针可广泛适用于在活体内探测众多的由pH值调控的蛋白-蛋白相互作用.
pH is a key factor that affects almost all surface charge distributions and related structural changes of all protein molecules, and the interaction between many protein molecules is also regulated by pH. In recent years, photo-crosslinking probes based on unnatural amino acids have been widely used Is used to capture protein-protein interactions in living cells.However, due to the changes of environmental pH often lead to significant changes in protein molecular structure, charging properties, existing non-natural amino acid photo-crosslinking probes are difficult to achieve in extreme pH Value of the interaction of the protein molecules capture and research.In this paper, the group will introduce the newly developed alkyl bis-aziridine active group-based non-natural amino acid light-crosslinking probe-DIZPK, through this probe , We successfully captured the role of HdeA, an important acidic molecular chaperone in Escherichia coli, during acid-stress in the membrane interstitial.In the captured HdeA substrate, we found two important molecules in membrane interstitium Chaperone proteins: DegP and SurA. By experiments we demonstrated that DegP and SurA can be protected by HdeA from forming aggregates under acidic stress conditions and subsequently Of the neutral process can assist HdeA in the refolding of other substrates.This ATP-independent molecular chaperone collaboration model may play a role in helping intestinal bacteria against acid stress.Based on the above experimental results, we propose A model of “molecular chaperone synergy” is used to explain the mechanism by which bacteria use antacid chaperones to increase their escape under acid stress. By extension, the introduction of highly adaptable sites in prokaryotes and eukaryotes Of the unnatural amino acid photo-cross-linked probes are widely applicable to the detection of numerous pH-regulated protein-protein interactions in vivo.