Aromatic Residues Display an Energetic Depth-Dependence in Lipid Bilayers that can be Modulated by Nearest-Neighbor Interactions

Abstract

The interfacial region of the bilayer is a complex environment composed of lipid head groups, phosphates, diglycerides and a gradient of water molecule density. Due to the statistical preference of the aromatic residues for this area of the bilayer, it is generally believed that these residues are important for both folding and positioning of proteins within the membrane. To interrogate if this preference has a thermodynamic basis, we measured the water-to-lipid free energy changes for the aromatic side chains as a function of depth in the lipid bilayer. We used outer membrane phospholipase A (OmpLA) as a host membrane protein to introduce lipid-facing amino acid substitutions. At twelve sites, we mutated host side chains to alanine, tryptophan, tyrosine and phenylalanine. Chemical denaturation and thermodynamic linkage were employed to measure side chain water-to-lipid free energy changes. These quantities were calculated by taking the difference between stabilities (ΔΔGow,l) of OmpLA aromatic variants from that of alanine, which was used as a reference state. The experimental data can be interpreted as a sum of water-to-lipid transfer free energies and specific nearest neighbor interactions between certain side chains. We used molecular dynamics simulations and double variant cycles to model and quantify these nearest neighbor interactions. Altogether, our findings indicate that the tryptophan and tyrosine aromatic side chains exhibit a depth-dependence in ΔΔGow,l values whereas phenylalanine shows no such trend. These data demonstrate that the enrichment of tryptophan and tyrosine in interfacial regions of bilayers is due to their favorable contributions to the thermodynamic stabilities of membrane proteins.