Juxtamembrane tryptophans have distinct roles in defining the OmpX barrel-micelle boundary and facilitating protein-micelle association.

Defining the span of the transmembrane region, a key requirement to ensure correct folding, stability and function of bacterial outer membrane β-barrels, is assisted by the amphipathic property of tryptophan. We demonstrate the unique and distinctive properties of the interface Trp76 and Trp140 of outer membrane protein X, and map their positional relevance to the refolding process, barrel formation and the resulting stability in dodecylphosphocholine micelles. The solvent-exposed Trp76 displays a rigid interfacial localization, whereas Trp140 is relatively micelle-solvated and contributes to barrel folding and global OmpX stability. Kinetic contribution to OmpX stability is influenced by the two tryptophans. Differential associations of the indoles with the detergent milieu therefore contribute to micelle-assisted β-barrel folding and concomitant OmpX stability.