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Use of Thiol-Disulfide Equilibria to Measure the Energetics of Assembly of Transmembrane Helices in Phospholipid Bilayers

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Use of Thiol-Disulfide Equilibria to Measure the Energetics of Assembly of Transmembrane Helices in Phospholipid Bilayers Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers Lidia Cristian, James D. Lear†, and William F. DeGrado† Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6059 Communicated by James A. Wells, Sunesis Pharmaceuticals, Inc., South San Francisco, CA, October 17, 2003 (received for review May 25, 2003) Despite significant efforts and promising progress, the under- association in detergent micelles (16). The reversible association standing of membrane protein folding lags behind that of soluble of a transmembrane peptide in micelles was measured by quan- proteins. Insights into the energetics of membrane protein folding titatively assessing the extent of disulfide formation under have been gained from biophysical studies in membrane-mimick- reversible redox conditions with a thiol-disulfide buffer. Here, ing environments (primarily detergent micelles). However, the we describe the application of the disulfide-coupled folding development of techniques for studying the thermodynamics of method to measure the energetics of transmembrane peptide folding in phospholipid bilayers remains a considerable challenge. association in phospholipid bilayers. The 19–46 transmembrane We had previously used thiol-disulfide exchange to study the fragment of the M2 protein from influenza A virus (M2TM19–46) thermodynamics of association of transmembrane ␣-helices in was used as a model membrane protein for this study. M2 is a detergent micelles; here, we extend this methodology to phos- small homotetrameric proton channel, consisting of 97-residue pholipid bilayers. The system for this study is the homotetrameric monomers (17–19). The protein has two cysteine residues at M2 proton channel protein from the influenza A virus. Transmem- positions 17 and 19 at the extracellular domain, which form a brane peptides from this protein specifically self-assemble into mixture of covalent dimers and tetramers (17). The active tetramers that retain the ability to bind to the drug amantadine. oligomeric form of M2 is homotetrameric (20). Proton channel Thiol-disulfide exchange under equilibrium conditions was used to activity has been reported in lipid bilayers from a synthetic quantitatively measure the thermodynamics of this folding inter- protein containing the predicted transmembrane region of M2 action in phospholipid bilayers. The effects of phospholipid acyl (21), suggesting that the channel-forming properties of the chain length and cholesterol on the peptide association were full-length protein reside in its transmembrane region. CD and investigated. The association of the helices strongly depends on solid-state NMR studies showed that the transmembrane seg- the thickness of the bilayer and cholesterol levels present in the ment of M2 adopts an ␣-helical structure in bilayers (22, 23), is phospholipid bilayer. The most favorable folding occurred when oligomeric in bilayers (24), and reversibly associates into tet- there was a good match between the width of the apolar region ramers in detergent micelles (16, 25, 26). A peptide from residues of the bilayer and the hydrophobic length of the transmembrane 19–46 contains a single native Cys residue, which reversibly helix. Physiologically relevant variations in the cholesterol level are forms intermolecular disulfides in the tetramer (16). Hence, this sufficient to strongly influence the association. Evaluation of the peptide has proven to be a very good candidate for thermody- energetics of peptide association in the presence and absence of cholesterol showed a significantly tighter association upon inclu- namic studies in micelles by thiol-disulfide interchange. Here, we sion of cholesterol in the lipid bilayers. investigate the ability of this peptide to associate in phospholipid bilayers and determine the effects of membrane thickness and added cholesterol on the equilibrium of association. n contrast to the substantial literature dealing with the struc- Itural energetics of water-soluble proteins, relatively little is Materials and Methods known about the forces that determine the stability of membrane Peptide Synthesis and Sample Preparation. M2TM19–46 peptide was proteins (1). The understanding of helical membrane protein synthesized and purified as described (16). Small unilamellar folding has been complicated by the difficulties associated with ͞ vesicles were prepared by codissolving M2TM19–46 trifluoro- structure determination and thermodynamic characterization of ethanol stock solutions with the appropriate amount of phos- membrane proteins. Compared with water-soluble proteins, pholipid from a stock solution in ethanol. The solvent was relatively few membrane protein structures are known at atomic evaporated under a stream of nitrogen, and the protein͞ resolution, although new structures are beginning to appear phospholipid film was kept overnight under high vacuum to more rapidly (1–8). Thermodynamic analysis of membrane remove all traces of solvent. The dry peptide͞phospholipid films proteins has been hampered by the experimental difficulties obtained were then hydrated in buffer (0.1 M Tris⅐HCl͞0.2 M imposed by their insolubility in water and great stability in KCl͞1 mM EDTA, pH 8.6), vortexed, and sonicated to clarity by membranes. Thus, to understand folding of membrane proteins using a bath sonicator (Laboratory Supplies, Hicksville, NY). it is essential to discover systems in which folding is in thermo- The concentration of peptide (20 ␮M) was kept constant while dynamic equilibrium and to develop methods to quantitatively varying the phospholipid concentration to attain the desired assess this equilibrium in membrane-like environments. Several ͞ biophysical techniques have been intensively used in thermody- peptide phospholipid mole ratios (typically between 1:100 and namic studies of membrane protein association in detergent 1:1,500). The phospholipids used in this study were: 1-palmitoyl- systems (9–15). However, the ideal environment for studying 2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dilauroyl-sn- membrane proteins is the lipid bilayer because lipids mimic more closely the native membrane environment. Thus, there is con- Abbreviations: POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DLPC, 1,2- siderable interest in finding reversible conditions under which dilauroyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocho- the thermodynamics of membrane protein association can be line; GSH, glutathione; GSSG, oxidized glutathione. studied in these environments. †To whom correspondence should be addressed. E-mail: [email protected] or Recently, we have reported a disulfide-coupled folding ap- [email protected]. proach to measure the energetics of transmembrane protein © 2003 by The National Academy of Sciences of the USA 14772–14777 ͉ PNAS ͉ December 9, 2003 ͉ vol. 100 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536751100 Downloaded by guest on October 1, 2021 Scheme 1. Thermodynamic coupling between disulfide formation and tetramerization. glycero-3-phosphocholine (DLPC), and 1,2-dimyristoyl-sn- Ellman’s reagent, 5,5Ј-dithiobis(2-nitrobenzoic acid). The glycero-3-phosphocholine (DMPC). All lipids were purchased amount of covalent dimer was calculated from the integrated from Avanti Polar Lipids. HPLC peak areas of the present species in the chromatograms Incorporation of amantadine into the peptide͞phospholipid by using the software supplied with the HPLC. samples was carried out by first adding the desired amount of For cholesterol concentration-dependence experiments, the amantadine from a trifluoroethanol stock solution to a glass vial peptide was incorporated into DLPC bilayers containing various and evaporating the solvent under a stream of nitrogen. The amounts of cholesterol. The peptide͞DPLC mole ratio was peptide was then incorporated into DLPC at a peptide͞ 1:500, and the final cholesterol concentration in the samples phospholipid mole ratio of 1:1,500 as described above; the ranged between 1 and 25 mol percent (relative to lipid concen- samples were hydrated in buffer, sonicated to clarity, and then tration). The ratio of GSSG to reduced GSH was 0.25. added to the amantadine films. Reversible disulfide formation was initiated by adding oxidized glutathione (GSSG) and re- Thermodynamic Analysis. The data obtained from thiol-disulfide duced glutathione (GSH) at varying ratios to the samples. The exchange measurements (see Figs. 3 and 5) were analyzed final molar ratios of peptide͞DLPC͞amantadine were 1:1,500:5, according to Scheme 1, which illustrates the thermodynamic 1:1,500:15, and 1:1,500:50. coupling between disulfide formation and tetramerization. This For samples containing cholesterol, mixtures of peptide, lipid, model is a simplified version of the more complex equilibria and the desired mol percentage of cholesterol (relative to lipid scheme we used previously (16). concentration) were codissolved from trifluoroethanol or etha- In Scheme 1, the monomeric and tetrameric states are given nol stock solutions, dried under nitrogen, and kept under high as M and T, respectively, and their oxidation states are indicated vacuum overnight. The dry films were then hydrated in buffer as a subscript. The factor of 3 for the upper value of K2 is related and sonicated to clarity in a bath sonicator. to the fact that a given Cys has three potential partners in the fully reduced tetramer, but only
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