Proteins folded before transportation
Export of proteins across a cytoplasmic membrane is an essential function of all cells. For unfolded proteins, there is the well-studied Sec pathway. However, the complex twin arginine translocation (Tat) pathway involving folded proteins is far less well understood. The SMTAT (Single-molecule imaging of twin-arginine transporter assembly) project used their newly developed artificial lipid bilayer to study the Tat pathway. With exceptional stability as well as simple reconstitution of membrane proteins, the model membrane enables single-molecule fluorescence imaging and single-channel electrical recording via gigaohm seals. Assembly of multiple copies of the three major proteins – TatA, TatB and TatC – to form a receptor complex and a translocase, enables transport of substrates of varying sizes. The SMTAT team evaluated the relative amounts of the three proteins in the complex. The protocol SMTAT devised to measure the stoichiometry of the individual proteins is a first and could be used as a basis for measuring other membrane proteins. The method used single molecule fluorescence and total internal reflection fluorescence (TIRF) microscopy. The chemical fluorophore at a substoichometric concentration was carefully photobleached to allow this process to proceed stepwise. To assemble an in vitro bilayer system with the reconstituted Tat proteins, the team incorporated all three in droplet interface bilayer systems. Their lateral diffusion constants were recorded after being observed as freely diffusing. In collaboration with other labs, SMTAT studied the interactions between Tat proteins at the molecular level. The work has resulted in a structural model for assembly of the active Tat translocase and confirmation that techniques such as molecule fluorescence and co-evolution analysis, based on multiple sequence alignments by sequence characters, can be used to predict protein interfaces in multi-subunit complexes. SMTAT research results have set the foundations for study of movement of proteins across the cytoplasmic membrane and applications would include drug design and study of progression of many diseases. The work has been published in eLife and Molecular Microbiology.
