Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy

Abstract

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. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the 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 ‘shield' that sequesters the IMP from water and promotes its solubilization.

A Water-Soluble DSBB Variant that Catalyzes Disulfide-Bond Formation In Vivo

“…Along similar lines, SIMPLExsolubilized human cyt b 5 stimulated the 17,20-lyase activity of CYP17A1 16 , an activity that is known to involve transmembrane helix-helix interactions between cyt b 5 and CYP17A1.…”

“…This feat was achieved by leveraging a method called SIMPLEx, which enables in vivo solubilization of IMPs in structurally and functionally relevant conformations without the need for potentially inactivating detergents, lipid reconstitutions, or mutations to the IMP itself 16 . The SIMPLEx technique was previously shown to be generally useful for solubilizing an array of structurally diverse IMPs, leading to the accumulation of nonaggregated, water-soluble IMPs at high titers (~5-10 mg/L of culture) that retained biological activity, namely ligand binding in the case of EmrE and stimulation of 17,20-lyase activity in the case of human cytochrome b 5 (cyt b 5 ) 16 . Here, we demonstrate for the first time in vivo catalysis with a SIMPLEx-solubilized enzyme in the context of a multienzyme pathway, opening the door to a wide array of in vivo applications involving solubilized IMPs.…”

“…1b). Previously, we showed that a truncation variant of human ApoAI lacking its 43-residue globular N-terminal domain (hereafter ApoAI*) promoted soluble expression of structurally diverse IMPs, which were recovered from the cytoplasm at high titers (~5-10 mg/L of culture) and in functionally relevant conformations 16 . Here, we designed chimeras in which the C-terminus of DsbB was genetically fused to ApoAI* while its N-terminus was fused to a highly soluble 'decoy' protein, namely the E. coli maltose-binding protein lacking its Nterminal export signal (cMBP, where c indicates cytoplasmic), to prevent the resulting chimera from becoming inserted in the cytoplasmic membrane (Fig.…”

“…1a) required a strategy for making the transmembrane protein DsbB compatible with the E. coli cytoplasm. To this end, we hypothesized that the SIMPLEx technique 16 could be used to create water-soluble DsbB variants that retain biocatalytic activity (i.e., re-oxidation of DsbA) in the cytoplasm (Fig. 1b).…”

“…When these solubilized DsbB chimeras were co-expressed with an exportdefective copy of DsbA, the normally periplasmic DsbB-DsbA pathway was completely recompartmentalized in the E. coli cytoplasm where it catalyzed de novo disulfide bond formation in a range of substrate proteins. While IMPs previously solubilized using SIMPLEx retained biological activity, as exemplified by ligand binding in the case of EmrE and stimulation of 17,20-lyase activity in the case of human cytochrome b 5 (cyt b 5 ) 16 , none of these were enzymes. Hence, the functional reconstitution of solubilized DsbB represents the first demonstration of catalytic activity in a solubilized IMP via SIMPLEx.…”