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

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 α-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.

[1]  D. Tieleman,et al.  Exploring models of the influenza A M2 channel: MD simulations in a phospholipid bilayer. , 2000, Biophysical journal.

[2]  Douglas C. Rees,et al.  The E. coli BtuCD Structure: A Framework for ABC Transporter Architecture and Mechanism , 2002, Science.

[3]  M. McNamee,et al.  Correlation between acetylcholine receptor function and structural properties of membranes. , 1986, Biochemistry.

[4]  T. Creighton An empirical approach to protein conformation stability and flexibility , 1983, Biopolymers.

[5]  G. Kochendoerfer,et al.  Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly. , 1999, Biochemistry.

[6]  Pavel Strop,et al.  Crystal Structure of Escherichia coli MscS, a Voltage-Modulated and Mechanosensitive Channel , 2002, Science.

[7]  D Needham,et al.  Elastic deformation and failure of lipid bilayer membranes containing cholesterol. , 1990, Biophysical journal.

[8]  W. DeGrado,et al.  Determination of membrane protein stability via thermodynamic coupling of folding to thiol–disulfide interchange , 2003, Protein science : a publication of the Protein Society.

[9]  W. DeGrado,et al.  Polar side chains drive the association of model transmembrane peptides. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[10]  I. Levitan,et al.  Modulation of endothelial inward-rectifier K+ current by optical isomers of cholesterol. , 2002, Biophysical journal.

[11]  R. Lamb,et al.  The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Eaton,et al.  Cholesterol in signal transduction. , 2000, Current opinion in cell biology.

[13]  Deborah A. Brown,et al.  Lipid-dependent Targeting of G Proteins into Rafts* , 2000, The Journal of Biological Chemistry.

[14]  R. Dutzler,et al.  X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity , 2002, Nature.

[15]  Anthony G. Lee,et al.  How lipids interact with an intrinsic membrane protein: the case of the calcium pump. , 1998, Biochimica et biophysica acta.

[16]  R. Mason,et al.  Attenuation of channel kinetics and conductance by cholesterol: An interpretation using structural stress as a unifying concept , 2004, The Journal of Membrane Biology.

[17]  M. Caffrey,et al.  Fluorescence quenching in model membranes. 3. Relationship between calcium adenosinetriphosphatase enzyme activity and the affinity of the protein for phosphatidylcholines with different acyl chain characteristics. , 1981, Biochemistry.

[18]  J. Killian,et al.  Hydrophobic mismatch between proteins and lipids in membranes. , 1998, Biochimica et biophysica acta.

[19]  Robert S. McDowell,et al.  A Minimal Peptide Scaffold for β-Turn Display: Optimizing a Strand Position in Disulfide-Cyclized β-Hairpins , 2001 .

[20]  T. Cross,et al.  Transmembrane four-helix bundle of influenza A M2 protein channel: structural implications from helix tilt and orientation. , 1997, Biophysical journal.

[21]  S. Regen Lipid-lipid recognition in fluid bilayers: solving the cholesterol mystery. , 2002, Current opinion in chemical biology.

[22]  N. Skelton,et al.  Turn stability in β‐hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns , 2003, Protein science : a publication of the Protein Society.

[23]  A. Lee,et al.  Interactions of cholesterol hemisuccinate with phospholipids and (Ca2+-Mg2+)-ATPase. , 1984, Biochemistry.

[24]  D. Schubert,et al.  Band 3 protein—cholesterol interactions in erythrocyte membranes , 1982, FEBS letters.

[25]  D. Rice,et al.  Effects of cholesterol on sodium-potassium ATPase ATP hydrolyzing activity in bovine kidney , 1988 .

[26]  D. Engelman,et al.  Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. , 1983, Journal of molecular biology.

[27]  T. Haltia,et al.  Forces and factors that contribute to the structural stability of membrane proteins. , 1995, Biochimica et biophysica acta.

[28]  M. Bretscher,et al.  Cholesterol and the Golgi apparatus. , 1993, Science.

[29]  T. Creighton,et al.  Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. , 1984, Journal of molecular biology.

[30]  M. Sugahara,et al.  Selective sterol-phospholipid associations in fluid bilayers. , 2002, Journal of the American Chemical Society.

[31]  J Wang,et al.  Structure of the transmembrane region of the M2 protein H+ channel , 2001, Protein science : a publication of the Protein Society.

[32]  W. DeGrado,et al.  pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus. , 2000, Biochemistry.

[33]  J. Silvius,et al.  Competition between cholesterol and phosphatidylcholine for the hydrophobic surface of sarcoplasmic reticulum Ca2+-ATPase. , 1984, Biochemistry.

[34]  R. Lamb,et al.  Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. , 1991, Virology.

[35]  Deborah A. Brown,et al.  Structure and Function of Sphingolipid- and Cholesterol-rich Membrane Rafts* , 2000, The Journal of Biological Chemistry.

[36]  B. Bechinger,et al.  Alignment of lysine-anchored membrane peptides under conditions of hydrophobic mismatch: a CD, 15N and 31P solid-state NMR spectroscopy investigation. , 2000, Biochemistry.

[37]  F. Maxfield,et al.  Role of Membrane Organization and Membrane Domains in Endocytic Lipid Trafficking , 2000, Traffic.

[38]  S. Regen,et al.  Nearest-Neighbor Recognition in Phospholipid Membranes. , 1997, Chemical reviews.

[39]  J. Killian,et al.  Sensitivity of single membrane-spanning alpha-helical peptides to hydrophobic mismatch with a lipid bilayer: effects on backbone structure, orientation, and extent of membrane incorporation. , 2001, Biochemistry.

[40]  A. Carruthers,et al.  Effects of lipid environment on membrane transport: the human erythrocyte sugar transport protein/lipid bilayer system. , 1988, Annual review of physiology.

[41]  D. Engelman,et al.  Glycophorin A helical transmembrane domains dimerize in phospholipid bilayers: a resonance energy transfer study. , 1994, Biochemistry.

[42]  J. East,et al.  Effects of lipid fatty acyl chain structure on the activity of the (Ca2+ + Mg2+)-ATPase. , 1986, Biochimica et biophysica acta.

[43]  G. Gimpl,et al.  Cholesterol as modulator of receptor function. , 1997, Biochemistry.

[44]  S. White,et al.  Membrane protein folding and stability: physical principles. , 1999, Annual review of biophysics and biomolecular structure.

[45]  R. McElhaney,et al.  Physical studies of cholesterol-phospholipid interactions , 1996 .

[46]  J. Ren,et al.  Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length. , 1999, Biochemistry.

[47]  M. Bloom,et al.  Models of lipid-protein interactions in membranes. , 1993, Annual review of biophysics and biomolecular structure.

[48]  T. Creighton Disulfide bonds as probes of protein folding pathways. , 1986, Methods in enzymology.

[49]  W. DeGrado,et al.  Sequence determinants of the energetics of folding of a transmembrane four-helix-bundle protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[50]  W. DeGrado,et al.  How do helix–helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo‐oligomeric helical bundles , 2003, Protein science : a publication of the Protein Society.

[51]  D. Brown,et al.  Functions of lipid rafts in biological membranes. , 1998, Annual review of cell and developmental biology.

[52]  K. Jacobson,et al.  Looking at lipid rafts? , 1999, Trends in cell biology.

[53]  L. Regan,et al.  Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein , 1994, Protein science : a publication of the Protein Society.

[54]  B. Baird,et al.  How does the plasma membrane participate in cellular signaling by receptors for immunoglobulin E? , 1999, Biophysical chemistry.

[55]  C. Fielding,et al.  Intracellular cholesterol transport. , 1997, Journal of lipid research.

[56]  M. G. Oakley,et al.  Design and characterization of a heterodimeric coiled coil that forms exclusively with an antiparallel relative helix orientation. , 2001, Journal of the American Chemical Society.

[57]  J. Ren,et al.  Transmembrane orientation of hydrophobic alpha-helices is regulated both by the relationship of helix length to bilayer thickness and by the cholesterol concentration. , 1997, Biochemistry.

[58]  Petra Fromme,et al.  Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution , 2001, Nature.

[59]  E. Ikonen,et al.  How cells handle cholesterol. , 2000, Science.

[60]  E. Ikonen,et al.  Roles of lipid rafts in membrane transport. , 2001, Current opinion in cell biology.

[61]  P. Yeagle Cholesterol and the cell membrane. , 1985, Biochimica et biophysica acta.

[62]  R. Lamb,et al.  Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block , 1993, Journal of virology.

[63]  D. Engelman,et al.  The effect of point mutations on the free energy of transmembrane alpha-helix dimerization. , 1997, Journal of molecular biology.

[64]  M. Bloom,et al.  Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes. , 1992, Biophysical journal.

[65]  R. Ashley,et al.  The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers. , 1992, Virology.

[66]  Lukas K. Tamm,et al.  Structure of outer membrane protein A transmembrane domain by NMR spectroscopy , 2001, Nature Structural Biology.

[67]  William F. DeGrado,et al.  Asparagine-mediated self-association of a model transmembrane helix , 2000, Nature Structural Biology.

[68]  Lawrence H. Pinto,et al.  Influenza virus M2 protein has ion channel activity , 1992, Cell.

[69]  T. Werge,et al.  Cholesterol-induced protein sorting: an analysis of energetic feasibility. , 2003, Biophysical journal.

[70]  Q Zhong,et al.  Two possible conducting states of the influenza A virus M2 ion channel , 2000, FEBS letters.

[71]  J. Tocanne,et al.  Is the protein/lipid hydrophobic matching principle relevant to membrane organization and functions? , 1999, FEBS letters.

[72]  N. C. Price,et al.  The secondary structure of influenza A M2 transmembrane domain A circular dichroism study , 1992, FEBS letters.

[73]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[74]  L. Liscum,et al.  Intracellular cholesterol transport. , 1992, Journal of lipid research.

[75]  P. S. Kim,et al.  Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure. , 1989, Biochemistry.

[76]  R. Stroud,et al.  Site-directed ligand discovery. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[77]  D. Engelman,et al.  Detergents modulate dimerization, but not helicity, of the glycophorin A transmembrane domain. , 1999, Journal of molecular biology.

[78]  J. M. East,et al.  Hydrophobic Mismatch and the Incorporation of Peptides into Lipid Bilayers: A Possible Mechanism for Retention in the Golgi† , 1998 .

[79]  J. Denny,et al.  Helix tilt of the M2 transmembrane peptide from influenza A virus: an intrinsic property. , 2000, Journal of molecular biology.