Membrane complexome of Pseudomonas aeruginosa

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.