A kinetic analysis of the folding and unfolding of OmpA in urea and guanidinium chloride: single and parallel pathways.

The outer membrane protein OmpA from Escherichia coli can fold into lipid vesicles and surfactant micelles from the urea-denatured state. However, a complete kinetic description of the folding and unfolding of OmpA, which can provide the basis for subsequent protein engineering studies of the protein's folding pathway, is lacking. Here we use two different denaturants to probe the unfolding mechanism of OmpA in the presence of the surfactant octyl maltoside (OM). Unfolding of OmpA in the presence of micelles, achieved with the potent denaturant guanidinium chloride (GdmCl), leads to single-phase unfolding. In contrast, OmpA unfolds in urea only below OM's critical micelle concentration, and this occurs in different phases, which we attribute to the existence of states that have bound different amounts of surfactant, from completely "naked" to partly covered by surfactant. Multiple parallel refolding phases are attributed to different levels of collapse prior to folding. Kinetic results used to derive the stability of OmpA in surfactant, using either urea or GdmCl as the denaturing agent, give comparable results and indicate a minimalist three-state folding scheme involving denatured state D, folding intermediate I, and native state N. N and I are stabilized by 15.6 and 2.6 kcal/mol, respectively, relative to D. The periplasmic domain of OmpA does not contribute to stability in surfactant micelles. However, BBP, a minimalist transmembrane β-barrel version of OmpA with shortened loops, is destabilized by ~10 kcal/mol compared to OmpA, highlighting loop contributions to OmpA stability.