Abstract
Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the β-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the β-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by ∼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the β-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted.