Two-state folding of the outer membrane protein X into a lipid bilayer membrane

Folding and insertion of β-barrel membrane proteins into native membranes is efficiently catalyzed by β-barrel assembly machineries. Understanding this catalysis requires a detailed description of the corresponding uncatalyzed folding mechanisms, which however have so far remained largely unclear. Here, we resolve folding and membrane insertion of the E. coli outer membrane protein X (OmpX) into 1,2-didecanoyl-sn-glycero-3-phosphocholine (PC10:0) membranes at the atomic level. By combining four different experimental techniques, we correlate global folding kinetics with global and local hydrogen bond formation kinetics. Under a well-defined reaction condition, these processes follow single-exponential velocity laws, with rate constants identical within experimental error. The data thus establish at atomic resolution that OmpX folds and inserts into the lipid bilayer of PC10:0 liposomes by a two-state mechanism.