Abstract
The development of methods for reversibly folding membrane proteins in a two-state manner remains a considerable challenge for studies of membrane protein stability. In recent years, a variety of techniques have been established and studies of membrane protein folding thermodynamics in the native bilayer environments have become feasible. Here we present the thiol-disulfide exchange method, a promising experimental approach for investigating the thermodynamics of transmembrane (TM) helix–helix association in membrane-mimicking environments. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. This experimental method provides a broadly applicable tool for thermodynamic studies of folding, oligomerization, and helix–helix interactions of membrane proteins.
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References
You M, Li E, Hristova K (2005) FRET in liposomes: measurements of TM helix dimerization in the native bilayer environment. Anal Biochem 340:154–164
Duong MT, Jaszewski TM, MacKenzie KR (2007) Changes in apparent free energy of helix-helix dimerization in a biological membrane due to point mutations. J Mol Biol 371:422–434
Cristian L, Lear JD, DeGrado WF (2003) Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers. Proc Natl Acad Sci USA 100:14772–14777
Chen L, Novicky L, Hristova K (2010) Measuring the energetic of membrane protein dimerization in mammalian membranes. J Am Chem Soc 132:3628–3635
DeGrado WF, Gratkowski H, Lear JD (2003) How do helix-helix interactions help determine the folds of membrane proteins? Perspectives from the study of homo-oligomeric helical bundles. Protein Sci 12: 647–665
MacKenzie KR, Fleming KG (2008) Association energetics of membrane spanning α-helices. Curr Opin Struct Biol 18:412–419
Regan L, Rockwell A, Wasserman Z, DeGrado WF (1994) Disulfide crosslinks to probe the structure and flexibility of a designed four-helix bundle protein. Protein Sci 3: 2419–2427
Zhang Y, Kulp DW, Lear JD et al (2009) Experimental and computational evaluation of forces directing the association of transmembrane helices. J Am Chem Soc 131: 11341–11343
Cristian L, Lear JD, Degrado WF (2003) Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange. Protein Sci 12: 1732–1740
Stouffer AL, Ma C, Cristian L et al (2008) The interplay of functional tuning, drug resistance, and thermodynamic stability in the evolution of the M2 proton channel from the influenza A virus. Structure 16:1067–1076
North B, Cristian L, Fu Stowell X et al (2006) Characterization of a membrane protein folding motif, the Ser zipper, using designed peptides. J Mol Biol 359:930–939
Choma C, Gratkowski H, Lear JD, DeGrado WF (2000) Asparagine-mediated self-association of a model transmembrane helix. Nat Struct Biol 7:161–166
Walshaw J, Woolfson DN (2001) Socket: a program for identifying and analysing coiled-coil motifs within protein structures. J Mol Biol 307:1427–1450
Mason JM, Arndt KM (2004) Coiled coil domains: stability, specificity, and biological implications. Chembiochem 5:170–176
Yu YB (2002) Coiled-coils: stability, specificity, and drug delivery potential. Adv Drug Deliv Rev 54:1113–1129
Acharya A, Rishi V, Vinson C (2006) Stability of 100 homo and heterotypic coiled-coil a-a′ pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R). Biochemistry 45:11324–11332
Wagschal K, Tripet B, Lavigne P, Mant C et al (1999) The role of position a in determining the stability and oligomerization state of alpha-helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins. Protein Sci 8:2312–2329
Lamb RA, Zebedee SL, Richardson CD (1985) Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell 40:627–633
Holsinger LJ, Lamb RA (1991) Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology 183:32–43
Pinto LH, Holsinger LJ, Lamb RA (1992) Influenza virus M2 protein has ion channel activity. Cell 69:517–528
Wang C, Takeuchi K, Pinto LH, Lamb RA (1993) Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. J Virol 67:5585–5594
Ren J, Lew S, Wang J, London E (1999) Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length. Biochemistry 38: 5905–5912
Killian JA (1998) Hydrophobic mismatch between proteins and lipids in membranes. Biochim Biophys Acta 1376:401–416
Adamian L, Liang J (2002) Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers. Proteins: Struct Funct Genet 47: 209–218
Yin H, Slusky JS, Berger BW et al (2007) Computational design of peptides that target transmembrane helices. Science 315: 1817–1822
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Cristian, L., Zhang, Y. (2013). Use of Thiol-Disulfide Exchange Method to Study Transmembrane Peptide Association in Membrane Environments. In: Ghirlanda, G., Senes, A. (eds) Membrane Proteins. Methods in Molecular Biology, vol 1063. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-583-5_1
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DOI: https://doi.org/10.1007/978-1-62703-583-5_1
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