Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism

Unfolded outer membrane protein A (OmpA) of Escherichia coil spontaneously inserts and refolds into Lipid bilayers upon dilution of denaturing urea. I n the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable o f monitoring the translocation across lipid bilayers of fluorescence report er groups such as tryptophan in real time [Kleinschmidt, J. I-I., and Tamm, L. K. (1999) Biochemistry 38, 4996-5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilaye rs via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the foldi ng pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their ref olded state was indistinguishable from the native state of OmpA. TDFQ analy sis of the translocation across the Lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 degrees C, all Trps started f rom a far distance from the bilayer center and then gradually approached a distance of approximately 10 Angstrom from the bilayer center. In a narrow temperature range between 30 and 35 degrees C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. Whe n monitored at 40 degrees C, which resolved the last steps of OmpA refoldin g, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 Angstrom from the center after refolding was complete. In contrast Trp-7 approached the 10 Angstrom distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four beta-hairpins of the eight-stranded beta-barrel of OmpA were very similar t o one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate p rovide strong evidence for a synchronous translocation of all four beta-hai rpins of OmpA across the lipid bilayer and suggest that OmpA inserts and fo lds into lipid bilayers by a concerted mechanism.