利用光交联探针研究pH值调控的蛋白-蛋白相互作用

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.

Probing pH mediated protein-protein interactions via photocrosslinking

Diverse protein-protein interactions are mediated by pH,a key parameter in dictating the charge and structure of nearly all proteins.Although photocrosslinking is powerful in trapping transient protein interactions in living cells,the pH-dependent protein interactions are exceedingly difficult to capture,largely due to the significant change of the protein binding interfaces upon pH variations.Here,we report that our newly developed alkyl diazirine-based unnatural amino acid photocrosslinker-DIZPK is capable of detecting the in vivo substrates of HdeA,a major acid-protection chaperone.Among the identified substrates of HdeA,we found two important periplasmic chaperone proteins：DegP and SurA,that were protected by HdeA at a low pH,but subsequently assist the HdeA-mediated recovery of other client proteins.This ATP-independent chaperone cooperation may enhance the acid resistance of enteric bacteria.Based on these results,we proposed a ＂chaperone cooperation＂ model in order to illustrate how acid-protection chaperones are employed by enteric bacterial to cope with acid stress.The highly adaptable photocrosslinker presented here can be generally applicable for surveying various pH-dependent protein-protein interactions in prokaryotic and eukaryotic cells.