Lipid Bilayer Insertion of OmpA from E. coli Requires the Association of Neighboring β-Strands, Revealed by Intramolecular Site Directed Fluorescence Quenching

Abstract

We present the first detailed study on the formation of neighboring antiparallel β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-strand- and β-barrel- formation were investigated for the 8-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring β-strands, oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were either closer to the periplasmic turns, named cis-side of the strand, or closer to the outer loops, named trans-side of the strand. WnCm-OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin-label, which is a short-range fluorescence quencher. To monitor association of neighboring β-strands, proximity between fluorescent W and labeled C was determined in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the presence of a lipid membrane. Residues in the trans-side of strands β1, β2, and β3, represented by mutants W15C35 (β1β2, trans) and W57C35 (β3β2, trans) reached close proximity prior to residues in the N(β1)- and C(β8)-terminal strands examined for mutants W15C162 (β1β8, trans) and W7C170 (β1β8, cis). Tryptophan and cysteine converged slightly faster in W15C162 (β1β8, trans) than in W7C170 (β1β8, cis). The last folding step was observed for residues at the cis-ends of strands β1 and β2 for the mutant W7C43 (β1β2, cis). The data demonstrate that neighboring β-strands associate while they insert into the hydrophobic core of the lipid bilayer.