Lill, H. | Search bioRxiv

bioRxiv2022

Autoinsertion (LAiR): rapid functional reconstitution of integral membrane proteins into lipid bilayers

Functional investigation of purified integral membrane proteins (IMPs) is hampered by the need to insert these hydrophobic proteins from the detergent-solubilized state into liposomal membranes. Here we report reintegration of IMPs into a lipid environment within minutes, an order of magnitude faster than currently used standard techniques. The new approach yielded optimal results for IMPs solubilized in the detergent lauryl-maltose neopentyl glycol (LMNG) and is therefore termed LMNG Auto-insertion Reintegration (LAiR). LAiR displays superior performance to standard methods in terms of protein activity, long-term stability and proton tightness of proteoliposomes. LAiR reconstituted vectorial control of membrane-bound activity by the transmembrane ion motive force, a property particularly important in mitochondrial function, which was undetectable by standard reintegration methods. LAiR also preserved fragile IMP properties that are prone to disruption upon reintegration, including long-term multi-subunit integrity, inhibitor susceptibility, and higher-order oligomeric states. LAiR proved suitable for reintegration into liposomes as well as into surface-tethered membrane bilayers, and was compatible with IMPs and lipids from prokaryotic and eukaryotic sources. We anticipate a broad scope for LAiR as a powerful tool in fundamental research, pharmaceutical applications, and biotechnology.

biochemistry↗

bioRxiv2020

Rapid diffusion of large TatA complexes detected using single particle tracking microscopy

The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of most bacteria and archaea. TatA, which contains a single membrane-spanning helix, is believed to be responsible for the actual translocation. According to the prevalent model, multiple TatA subunits form a transient protein-conducting pore, which disassembles after each translocation event. An alternative model exists, in which TatA proteins locally weaken the lipid bilayer to translocate folded proteins. Here, we imaged eGFP-fused TatA expressed from the genome in live E. coli cells. Images showed TatA occuring both in highly mobile monomers or small oligomers and in large, stable complexes that do not dissociate. Single-particle tracking revealed that large TatA complexes switch between fast and slow diffusion. The fast diffusion is too fast for a transmembrane protein complex consisting of multiple TatA monomers. In line with recent data on rhomboid proteases, we propose that TatA complexes switch between a slowly diffusing transmembrane conformation and a rapidly diffusing membrane-disrupting state that enables folded proteins to cross the membrane, in accordance with the membrane-weakening model.

microbiology↗