Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers

Abstract

Despite significant efforts and promising progress, the understanding of membrane protein folding lags behind that of soluble proteins. Insights into the energetics of membrane protein folding have been gained from biophysical studies in membrane-mimicking environments (primarily detergent micelles). However, the development of techniques for studying the thermodynamics of folding in phospholipid bilayers remains a considerable challenge. We had previously used thiol-disulfide exchange to study the thermodynamics of association of transmembrane ␣-helices in detergent micelles; here, we extend this methodology to phospholipid bilayers. The system for this study is the homotetrameric M2 proton channel protein from the influenza A virus. Transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. Thiol-disulfide exchange under equilibrium conditions was used to quantitatively measure the thermodynamics of this folding interaction in phospholipid bilayers. The effects of phospholipid acyl chain length and cholesterol on the peptide association were investigated. The association of the helices strongly depends on the thickness of the bilayer and cholesterol levels present in the phospholipid bilayer. The most favorable folding occurred when there was a good match between the width of the apolar region of the bilayer and the hydrophobic length of the transmembrane helix. Physiologically relevant variations in the cholesterol level are sufficient to strongly influence the association. Evaluation of the energetics of peptide association in the presence and absence of cholesterol showed a significantly tighter association upon inclusion of cholesterol in the lipid bilayers.

Proton and cation transport activity of the M2 proton channel from influenza A virus

“…The improved reconstitution methods used herein gave M2PLs with the protein unidirectionally inserted as in the native virus. Cholesterol is important for maximal activity and efficient reconstitution, which is consistent with its known interaction with cholesterol (44) (45). The proper insertion might also be related to the curvature of the vesicles, because a cytoplasmic helix immediately C-terminal to the TM helix of M2 plays an important role in cholesterol-binding and determining the morphology of the virus (46).…”

Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel

“…Furthermore, the data in Fig. 5 can be used to assess ranges of peptide/detergent ratios over which the peptide is predominantly tetrameric at a given pH; also, because A/M2 is far more stable in bilayers than in micelles (32), these measurements place lower limits on the stability in vesicles and multibilayer environments.…”

Changes in Apparent Free Energy of Helix–Helix Dimerization in a Biological Membrane Due to Point Mutations

“…Assuming that the fusion protein is present in the cell membrane only as monomers or dimers, the oligomerization reaction of the fusion protein and the corresponding equilibrium association constant K a are given by (1) where [P] represents the mole fraction of monomeric fusion protein (relative to moles of all lipids and proteins in the membrane), [P 2 ] represents the mole fraction of dimeric fusion protein, and the mole fraction of total protein, [P total ], is given by (2) and so (3) In the limit where [P 2 ] is small we can make the approximation (4) which simplifies the expression for the apparent equilibrium constant to (5) This expression for K app can be related to measurable parameters from the TOXCAT assay. We assume that the mole fraction of dimeric TOXCAT fusion protein, [P 2 ], is given by the expression level of the reporter gene, CAT lysate , times a proportionality constant α, (6) and that the mole fraction of total TOXCAT fusion protein, [P total ], is given by the western blot band intensity of a standardized amount of membrane, WB lysate , times a proportionality constant β that includes the moles of total lipid and protein in the membrane aliquot (7) To determine the absolute K app (and thus the apparent free energy of association, ΔΔG app ), we would need the proportionality constants α and β. However, when we write an expression for the ratio of the apparent association constants for wild type and mutant TMDs (8) we can rearrange the terms, cancel the proportionality constants, and calculate ΔΔG app , the apparent change in free energy of association that results from a mutation to the TMD sequence, using the expression (9) where the term CAT mut /CAT WT is the ratio of CAT activity from normalized amounts of cells expressing mutant or wild type constructs, and the term WB WT /WB mut is the ratio of the band intensities for wild type or mutant TOXCAT fusion proteins on a western blot of normalized amounts of cells.…”