Potential of Mean Force and pK_a Profile Calculation for a Lipid Membrane-Exposed Arginine Side Chain

Abstract

The issue of ionizable protein side chains interacting with lipid membranes has been the focus of much attention since the proposal of the paddle model of voltage-gated ion channels, which suggested multiple arginine (Arg) side chains may move through the hydrocarbon core of a lipid membrane. Recent cell biology experiments have also been interpreted to suggest that these side chains would face only small free energy penalties to cross membranes, challenging a long-standing view in membrane biophysics. Here, we employ side chain analog and transmembrane helix models to determine the free energy of an Arg side chain, as a function of protonation state, across a membrane. We observe high free energy barriers for both the charged and neutral states that would prohibit lipid-exposed movement. The mechanisms for charged and neutral Arg transport are, however, very different, with the neutral state experiencing simple dehydration, whereas the charged state experiences a complex mechanism involving connections to the bilayer interfaces that deform the local membrane structure. We employ special methods to ensure sampling of these interfacial connections and decompose the free energy to shed light on the mechanisms. These deformations are found to preferentially stabilize the protonated form, such that the Arg side chain remains almost exclusively charged inside the membrane, with a pKa shift of <or=4.5 units. In contrast, the analog models are found to exaggerate the variations in energetics across the membrane and have larger pKa shifts. These results have implications for models of voltage gated ion channels, suggesting that although Arg side chains are ideally suited for carrying charge, the thermodynamics dictate that they must remain sequestered from the lipid bilayer environment.

A Conserved Asparagine Residue in Transmembrane Segment 1 (TM1) of Serotonin Transporter Dictates Chloride-coupled Neurotransmitter Transport

“…These findings are consistent with the proposition that Cys-336 acts similar to charge-changing (Asp or Glu) mutations at the position 372 in providing a localized negative charge and thereby results in Cl Ϫ independence. Whereas the convergence of free energy simulations is known to be a bottleneck of the method, large shifts in pK a values are suggestive of different propensities in the protonation states of these two cysteines (67).…”

Exploring the nature of the translocon-assisted protein insertion

“…The original implication has been that the translocon effect is relatively small and that the experimental findings are related to the energetics of the specific amino acid in the center of the membrane. This issue is important in view of attempts to point out the possibility that the experiments have not established that the controversial low ΔG app for a positively charged Arg corresponds to Arg in the center of the membrane, since we have an obvious possibility that the Arg side chain is tilted toward the membrane surface and that this explains the low ΔG app (9,10,17). We point out in this respect that one can use a more trivial suggestion, just saying that the center of the helix can move and place the central charge closer to the membrane surface.…”

On the energetics of translocon-assisted insertion of charged transmembrane helices into membranes

“…In particular, one would like to understand the reason for the small values of the free energy associated with the insertion of charged residues. This presents a significant problem, as the free energy of placing a charge in a nonpolar environment is expected to be very large (11), and because some attempts to simulate the relevant free energy penalty lead in most cases to a significantly larger estimate of the ΔG app (by more than 10 kcal∕mol) (12,13) than the observed one (2-4 kcal∕mol) (5). Different justifications have been given to the observed values, including the effect of water penetration, the effects of the lipid head groups, position of the charge in the membrane, and even membrane distortion (13).…”