Side-chain hydrophobicity scale derived from transmembrane protein folding into lipid bilayers

Abstract

The transfer free energies of the twenty natural amino acid side chains from water to phospholipid bilayers make a major contribution to the assembly and function of membrane proteins. Measurements of those transfer free energies will facilitate the identification of membrane protein sequences and aid in the understanding of how proteins interact with membranes during key biological events. We report the first water-to-bilayer transfer free energy scale (i.e., a "hydrophobicity scale") for the twenty natural amino acid side chains measured in the context of a native transmembrane protein and a phospholipid bilayer. Our measurements reveal parity for apolar side-chain contributions between soluble and membrane proteins and further demonstrate that an arginine side-chain placed near the middle of a lipid bilayer is accommodated with much less energetic cost than predicted by molecular dynamics simulations.

Fig. 1.

Outer membrane phospholipase A (OmpLA) as a host-guest system for measuring the membrane insertion energies of lipid-exposed amino acid residues. (A) Backbone of OmpLA rendered using PyMol (DeLano Scientific) and Protein Data Bank (PDB) ID code 1qd5 with the α-carbon of the alanine at sequence position 210 shown as a black sphere. Solid horizontal lines represent the boundaries of OmpLA’s transmembrane region. The dashed horizontal line represents the midplane of that region. (B–E) Example denaturation data for A210X sequence variants, each with a fit line from a three-state linear-extrapolation model. Data points represent tryptophan fluorescence emission measurements, normalized such that the emission from folded protein is 1 and the emission from unfolded protein is 0. Data for the wild-type OmpLA is shown in black in each panel. (B) Data for X = F, I, L, M, P, V, W, and Y are shown in orange. (C) Data for X = C and G are shown in gray. (D) Data for X = N, Q, S, and T are shown in teal. (E) Data for X = D, E, H, K, and R are shown in blue.

Fig. 2.

Whole-protein hydrophobicity scale determined from the OmpLA system. The difference in the Gibbs free energy of unfolding () of each amino acid variant at position 210 is compared to the wild-type OmpLA. Error bars represent standard errors of the mean from individual titration experiments. The color scheme is the same as in Fig. 1 B–E.

Fig. 3.

Energetics of side-chain partitioning varies by depth in the membrane. The OmpLA host-guest system is shown similarly as in Fig. 1 with the α-carbons of sequence positions 120, 164, 210, 212, 214, and 223 shown as black spheres. The membrane depth of those five α-carbons versus the of leucine and arginine variants (compared to alanine variants) is shown aligned with the OmpLA image. Normal distributions fit to the leucine and arginine data are also shown. Error bars represent standard errors of the mean from individual titration experiments.

Fig. 4.

A double-mutant cycle reveals that two arginines cooperate energetically to partition into a membrane. The OmpLA system is shown similarly as in Fig. 1 with the atoms of the side chains varied in the cycle shown as spheres. The arginine side-chain orientations shown are for illustration of size and are not intended to depict any known side-chain conformations relative to the lipid bilayer in our experiments.