Thermodynamic partitioning forces at the membrane protein interface

Abstract

Transmembrane proteins fold by the orchestrated interplay of membrane chaperones, holdases and the lipid membrane. Upon folding, membrane protein systems attain a thermodynamically stabilized framework through optimized energetics involving protein – membrane – solvent interactions. In particular, the water-bilayer interface demands selectively positioned amino acids to arbitrate interactions between the hydrophobic interior and polar phospholipid headgroups. A fundamental question that arises is the extent to which the amino acid side chain contributes to membrane protein folding and in maintaining protein-lipid interactions at the intricate interface environment of membrane proteins. Our goal is to experimentally deduce the partitioning cost of amino acids at the interface, and the associated physical principles governing the overall scaffold stability. Interface residues are important contributors for membrane protein folding, and additionally serve as post-folding membrane anchors. In this study, we have quantified the free energy change associated with the chemical nature of the residue at the membrane interface. We achieve this by studying the unassisted folding equilibrium of the 8-stranded transmembrane β-barrel enzyme PagP in phosphocholine lipidic micelles and lipid vesicles. We present the first experimentally measured whole-protein free energy scale for side chain partitioning at the membrane interface, for all naturally abundant amino acids. We obtain differential contributions of the protein- and lipid-facing interface residues to the barrel folding pathway and stability of the folded scaffold. The most favorable transfer at the amphiphilic lipid-facing interface is for hydrophobic amino acids, whereas we observe the highest energetic cost of transfer for charged and polar groups. On the contrary, we find that small and polar residues are most favorably transferred to the protein-facing interface, whereas hydrophobic and aromatic residues promote alternate folding pathways in PagP. Our studies establish that the side chain non-polar accessible surface area shows a direct correlation with the partitioning free energy change at the lipid-facing interface residue. We also find that the non-polar accessible surface correlates inversely with the thermodynamic partitioning free energy change when the interface residue is protein-facing. We demonstrate how PagP maintains a balance between concerted folding and hydrophobic collapse by evolutionary choice of small polar residues at the protein-facing interface. We also identify the importance of interface polar residues to the folding pathway of β-barrel membrane proteins.