AIChE Annual Meeting
Wednesday, November 1, 2017 - 2:55pm to 3:00pm
Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. As an alternative to extraction and in vitro solubilization, we devised a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic âshieldâ that sequesters the IMP from water and promotes its solubilization. In one notable application, we converted Escherichia coli DsbB, a transmembrane enzyme that catalyzes the re-oxidation of the periplasmic oxidase DsbA by ubiquinone, into a water-soluble biocatalyst using SIMPLEx. When solubilized DsbB variants were co-expressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide bond formation in a range of substrate proteins and in a manner that depended on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, the generality of the solubilization technique should pave the way for unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid bilayer context.