Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site-directed fluorescence quenching

We present a detailed study on the formation of neighboring β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-Strand and β-barrel formations were investigated for the eight-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 closer to either the periplasmic turns (named cis-side) or 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 the association of neighboring β-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the 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 as 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 also demonstrate that the neighboring β-strands associate upon insertion into the hydrophobic core of the lipid bilayer