Reversible folding energetics of Yersinia Ail barrel reveals a hyperfluorescent intermediate.

Deducing the molecular details of membrane protein folding has lately become an important area of research in biology. Using Ail, an outer membrane protein (OMP) from Yersina pestis as our model, we explore details of β-barrel folding, stability, and unfolding. Ail displays a simple transmembrane β-barrel topology. Here, we find that Ail follows a simple two-state mechanism in its folding and unfolding thermodynamics. Interestingly, Ail displays multi-step folding kinetics. The early kinetic intermediates in the folding pathway populate near the unfolded state (βT ≈ 0.20), and do not display detectable changes in the local environment of the two interface indoles. Interestingly, tryptophans regulate the late events of barrel rearrangement, and Ail thermodynamic stability. We show that W149 → Y/F/A substitution destabilizes Ail by ~0.13-1.7 kcal mol-1, but retains path-independent thermodynamic equilibrium of Ail. In surprising contrast, substituting W42 and retaining W149 shifts the thermodynamic equilibrium to an apparent kinetic retardation of only the unfolding process, which gives rise to an associated increase in scaffold stability by ~0.3-1.1 kcal mol-1. This is accompanied by the formation of an unusual hyperfluorescent state in the unfolding pathway that is more structured, and represents a conformationally dynamic unfolding intermediate with the interface W149 now lipid solvated. The defined role of each tryptophan and poorer folding efficiency of Trp mutants together presents compelling evidence for the importance of interface aromatics in the unique (un)folding pathway of Ail, and offers interesting insight on alternative pathways in generalized OMP assembly and unfolding mechanisms.