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.T he transfer of amino acid side chains from water into phospholipid lipid bilayers is a fundamental energetic contribution to the total thermodynamic stability of transmembrane proteins (1, 2). A hydrophobicity scale that ranks the water-to-bilayer transfer energies of the twenty natural amino acid side chains could allow the identification of genes that code for membrane proteins (3-6) and aid the understanding of how proteins interact with membranes during key biological events, such as cell signaling, drug binding, and the gating of ion-channels. However, despite the abundance of available hydrophobicity scales, there have been no experimental measurements of water-to-bilayer transfer free energies of amino acid side chains that derive from the partitioning of a native transmembrane protein from water to the interior of a phospholipid bilayer. Existing estimations of water-to-bilayer partitioning of side chains were obtained from systems comprising small compounds to represent proteins and/or organic solvents to mimic bilayers. The only experimental hydrophobicity scale derived from proteins and bilayers (the "translocon scale") described the translocon-to-bilayer transition (7,8). Mediated membrane insertion by the translocon complex does not explain the stability of membrane proteins in lipid bilayers relative to a water-solvated state.Here we achieve thermodynamic measurements of amino acid side-chain transfer into lipid bilayers in the context of a natively folded transmembrane protein spanning a phospholipid bilayer. Our measurements reveal the hydrophobic effect contributes equivalently to the stability of both membrane and soluble proteins (9). Our experiments further demonstrate that a membrane protein can accommodate an arginine side chain placed near the apolar middle of a lipid bilayer with much less cost in energy than has been previously predicted by molecular dynamics simulations (10-11). Moreover, we found the membrane partitioning of two arginine residues to be thermodynamically cooperative. Therefore, our measurements should inform the study of voltage-sensing ion channels, which concertedly ...

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

“…The thermodynamic pull in this sorting hypothesis originates from the observations of unusually high OMP folding stabilities, DG Fold . OmpLA, PagP and OmpW all have free energies of folding into large unilamellar vesicles (LUVs) in the range 218 to 232 kcal mol 21 [9,10]. These are very large values, and an interesting thought experiment can be constructed to consider their magnitudes: if an Avogadro's number of OMPs possessing the stability of OmpLA (232 kcal mol 21 ) could be envisioned to exist within a Gram-negative bacterium, at equilibrium fewer than 100 of these molecules would be unfolded.…”

Buried lysine, but not arginine, titrates and alters transmembrane helix tilt

“…Additionally, for cases in which the aqueous access to Arg may be impeded, structural manipulations to remove the obstruction can restore the accommodation of Arg within a bilayer (19). In relatively thin dilauroyl-phosphatidylcholine bilayer membranes, the cost of placing Arg within a transmembrane helix of outer membrane phospholipase is about 3.7 kcal/mol at pH 3.8 (13,16,18), whereas a somewhat larger cost was found for placing Lys at the same position at pH 3.8 (13). Within the context of the existing experiments and simulations, the pH dependence of side-chain ionization merits consideration.…”

Temperature dependence of amino acid hydrophobicities

“…The water-to-cyclohexane distribution coefficients (K w>c ) of the 20 common sidechains [here termed "hydrophobicities" (6, 7) and expressed in concentration units of mol/L in each phase; SI Appendix] were found to span a range of 15 orders of magnitude at pH 7 and 25°C. Values of K w>c have been shown to be related to their outside-to-inside distributions in globular proteins (4,8) and to their tendencies to appear within the buried sequences of transmembrane proteins (9)(10)(11).…”