Interactome of NbdA containing nanodiscs

Studies of membrane protein-protein interactions are very important to fully understand the biological function of a cell. The solubilization of proteins from the native membrane environment is a critical step in the preparation of membrane proteins that might affect the stability of protein complexes. In this work, we used the amphiphilic diisobutylene / maleic acid copolymer (DIBMA) to create a soluble library of the membrane proteome of the opportunistic pathogen Pseudomonas aeruginosa. Size fractionation of polymer nanodisc-embedded proteins and subsequent mass spectrometry led to the identification of 3358 proteins. The library showed a very good overall coverage compared to previous proteome data. The pattern of size fractionation indicated the preservation of protein complexes in the library. More than 20 previously described complexes, e.g., the SecYEG and Pili complexes, were identified and analyzed for coelution. Although most protein complexes seem to be preserved in the library, the data set does not permit the de novo prediction of protein complexes. However, the experimental approach to identify protein interactions in the soluble library using pulldown assays in combination with mass spectrometry was successful. Employing the membrane phosphodiesterase NbdA, a member of the c-di-GMP network as a bait, 35 novel candidate proteins for interaction were identified. With the ATPase PilB, a novel interaction partner of NbdA was confirmed using the bacterial adenylate cyclase two hybrid assay. Taken together, this work describes the versatility of the soluble membrane proteome library of P. aeruginosa for the investigation of protein interactions and membrane protein complexes.膜蛋白-蛋白相互作用的研究对于全面阐明细胞的生物学功能具有至关重要的意义。将蛋白质从天然膜环境中增溶，是膜蛋白制备过程中的关键步骤，该过程可能会影响蛋白质复合物的稳定性。本研究采用两亲性二异丁烯/马来酸共聚物（DIBMA），构建了机会致病菌铜绿假单胞菌（Pseudomonas aeruginosa）膜蛋白质组的可溶性文库。通过对聚合物纳米圆盘包埋的蛋白质进行尺寸分级分离，并结合后续的质谱分析，共鉴定出3358种蛋白质。与既往蛋白质组数据相比，该文库展现出极佳的整体覆盖度。尺寸分级分离的结果表明，文库中的蛋白质复合物得以保留。研究鉴定并分析了20余种此前已报道的复合物，例如SecYEG复合物和菌毛复合物的共洗脱特性。尽管大多数蛋白质复合物似乎在该文库中得到了保留，但本数据集无法支持蛋白质复合物的从头预测。不过，通过结合下拉实验与质谱分析来鉴定可溶性文库中蛋白质相互作用的实验策略取得了成功。以环二鸟苷酸（c-di-GMP）网络成员、膜磷酸二酯酶NbdA作为诱饵蛋白，共鉴定出35种新型相互作用候选蛋白。通过细菌腺嘌呤环化酶双杂交实验，验证了ATP酶PilB是NbdA的新型相互作用伴侣。综上，本研究展示了铜绿假单胞菌膜蛋白质组可溶性文库在研究蛋白质相互作用与膜蛋白复合物方面的广泛适用性。