On the thermodynamic stability of a charged arginine side chain in a transmembrane helix

Abstract

Biological membranes consist of bilayer arrangements of lipids forming a hydrophobic core that presents a physical barrier to all polar and charged molecules. This long-held notion has recently been challenged by biological translocon-based experiments that report small apparent free energies to insert charged side chains near the center of a transmembrane (TM) helix. We have carried out fully atomistic simulations to provide the free-energy profile for moving a TM helix containing a protonated Arg side chain across a lipid bilayer. Our results reveal the fundamental thermodynamics governing the stability of charged side chains in membranes and the microscopic interactions involved. Despite local membrane deformations, where large amounts of water and lipid head groups are pulled into the bilayer to interact with Arg, the free-energy barrier is 17 kcal/mol. We provide a rationale for the differences in our microscopic free energies and cell biological experiments using free-energy calculations that indicate that a protonated Arg at the central residue of a TM helix of the Leader peptidase might reside close to the interface and not at the membrane center. Our findings have implications for the gating mechanisms of voltage-gated ion channels, suggesting that movements of protonated Arg residues through the membrane will be prohibited.

Fig. 1.

TM helix system of 31,593 atoms. An 80-residue polyLeu α-helix (green) with neutral N (bottom) and C (top) termini and a central Arg (gray/cyan/white) spans a dipalmitoylphosphatidylcholine bilayer with 0.5 M KCl baths, containing 7,895 water molecules (red/white sticks), 72 K⁺ (blue balls), and 73 Cl⁻ (gray balls) ions. Water and P atoms near Arg are shown as red/white and yellow balls, respectively.

Fig. 2.

PMF for the polyLeu+Arg TM helix (Lower), and MD snapshots when Arg is within the membrane core (Upper).

Fig. 3.

Free-energy contributions from water (red), head groups (blue), and ions (cyan) drawn into the bilayer core. The black curve is the total PMF, and the green curve is the remainder after subtracting core group contributions.

Fig. 4.

Side-chain structural isomers. (A) Adiabatic energy map for an Arg side chain. Low energy states are labeled a–h. (B) The structures in A are illustrated. (C) MD rotamer distributions for Arg in the bulk (|z| > 22 Å), lower interface (−22 ≤ z < −13 Å), upper interface (13 < z ≤ 22 Å), lower core (−13 ≤ z < −4 Å), central core (−4 ≤ z < 4 Å), and upper core (4 ≤ z < 13 Å).

Fig. 5.

2D PMF as a function of helix position (z) and relative side-chain–helix position, s = z_C − z, obtained from a combination of 1D and 2D (dashed-box region) biased simulations.

Fig. 6.

Relating to translocon experiments. (A) Sample background helix (Lep+Leu) MD configurations. (B) Background Lep PMF (blue curve), polyLeu+Arg PMF from Fig. 2 (dotted black curve), and total PMF (Lep+Arg) (red curve).