Insights into the transport mechanism of energy-coupling factor transporters

Changes in membrane proteins conformation can be problematic to study due to the lipid bilayer environment. It is getting even more challenging when the predicted movement is toppling, a rotation of the protein in the lipid bilayer. In Energy-coupling factor (ECF) transporters, the substrate-binding subunit (named S-component) is hypothesized to undergo toppling during the transport cycle. There is no direct observation of toppling reported until now. However, indirect results (crystal structures, MD simulations, competition assays) provide enough basis to search for direct confirmation of this protein movement. Here, we tried to apply three different methods to capture S-component toppling: cysteine crosslinking, use of environment sensitive dye (RH421), and FTIR spectroscopy. None of the methods provided a clear answer about toppling of S-components.