Advertisement

Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM™)

Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

A described simple and advanced protocol for the substituted-cysteine accessibility method as applied to transmembrane (TM) orientation (SCAM™) permits a topology analysis of proteins in their native state and can be universally adapted to any membrane system to either systematically map an uniform topology or identify and quantify the degree of mixed topology. In this approach, noncritical individual amino acids that are thought to reside in the putative extracellular or intracellular loops of a membrane protein are replaced one at a time by cysteine residue, and the orientation with respect to the membrane is evaluated using a pair of membrane-impermeable nondetectable and detectable thiol-reactive labeling reagents.

Key words

Membrane protein Topology Cysteine Maleimides SCAM™ 

References

  1. 1.
    von Heijne G (2006) Membrane-protein topology. Nat Rev Mol Cell Biol 7:909–918CrossRefGoogle Scholar
  2. 2.
    Bogdanov M, Xie J, Dowhan W (2009) Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule. J Biol Chem 284:9637–9641CrossRefGoogle Scholar
  3. 3.
    Dowhan W, Bogdanov M (2009) Lipid-dependent membrane protein topogenesis. Annu Rev Biochem 78:515–540CrossRefGoogle Scholar
  4. 4.
    Bogdanov M, Dowhan W, Vitrac H (2014) Lipids and topological rules governing membrane protein assembly. Biochim Biophys Acta 1843:1475–1488CrossRefGoogle Scholar
  5. 5.
    Bogdanov M, Zhang W, Xie J, Dowhan W (2005) Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM™): application to lipid-specific membrane protein topogenesis. Methods 36:148–171CrossRefGoogle Scholar
  6. 6.
    Fleishman SJ, Unger VM, Ben-Tal N (2006) Transmembrane protein structures without X-rays. Trends Biochem Sci 31:106–113CrossRefGoogle Scholar
  7. 7.
    Lacapere JJ, Pebay-Peyroula E, Neumann JM, Etchebest C (2007) Determining membrane protein structures: still a challenge! Trends Biochem Sci 32:259–270CrossRefGoogle Scholar
  8. 8.
    Bochud A, Ramachandra N, Conzelmann A (2013) Adaptation of low-resolution methods for the study of yeast microsomal polytopic membrane proteins: a methodological review. Biochem Soc Trans 41:35–42CrossRefGoogle Scholar
  9. 9.
    Bogdanov M, Heacock PN, Dowhan W (2010) Study of polytopic membrane protein topological organization as a function of membrane lipid composition. Methods Mol Biol 619:79–101CrossRefGoogle Scholar
  10. 10.
    Bogdanov M, Xie J, Heacock P, Dowhan W (2008) To flip or not to flip: lipid-protein charge interactions are a determinant of final membrane protein topology. J Cell Biol 182:925–935CrossRefGoogle Scholar
  11. 11.
    Nasie I, Steiner-Mordoch S, Gold A, Schuldiner S (2010) Topologically random insertion of EmrE supports a pathway for evolution of inverted repeats in ion-coupled transporters. J Biol Chem 285:15234–15244CrossRefGoogle Scholar
  12. 12.
    Zhu Q, Casey JR (2007) Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology. Methods 41:439–450CrossRefGoogle Scholar
  13. 13.
    Islam ST, Lam JS (2013) Topological mapping methods for alpha-helical bacterial membrane proteins--an update and a guide. Microbiology 2:350–364Google Scholar
  14. 14.
    Lee H, Kim H (2014) Membrane topology of transmembrane proteins: determinants and experimental tools. Biochem Biophys Res Commun 453:268–276CrossRefGoogle Scholar
  15. 15.
    Liapakis G (2014) Obtaining structural and functional information for GPCRs using the substituted-cysteine accessibility method (SCAM). Curr Pharm Biotechnol 15:980–986CrossRefGoogle Scholar
  16. 16.
    van Geest M, Lolkema JS (2000) Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev 64:13–33CrossRefGoogle Scholar
  17. 17.
    van Geest M, Lolkema JS (1999) Transmembrane segment (TMS) VIII of the Na(+)/citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane. J Biol Chem 274:29705–29711CrossRefGoogle Scholar
  18. 18.
    Karlin A, Akabas MH (1998) Substituted-cysteine accessibility method. Methods Enzymol 293:123–145CrossRefGoogle Scholar
  19. 19.
    Bogdanov M, Heacock PN, Dowhan W (2002) A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition. EMBO J 21:2107–2116CrossRefGoogle Scholar
  20. 20.
    Bogdanov M, Heacock P, Guan Z, Dowhan W (2010) Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli. Proc Natl Acad Sci U S A 107:15057–15062CrossRefGoogle Scholar
  21. 21.
    Bogdanov M, Dowhan W (2012) Lipid-dependent generation of a dual topology for a membrane protein. J Biol Chem 287:37939–37948CrossRefGoogle Scholar
  22. 22.
    Vitrac H, Bogdanov M, Heacock P, Dowhan W (2011) Lipids and topological rules of membrane protein assembly: balance between long- and short-range lipid-protein interactions. J Biol Chem 286:15182–15194CrossRefGoogle Scholar
  23. 23.
    Vitrac H, Bogdanov M, Dowhan W (2013) In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein. Proc Natl Acad Sci U S A 110:9338–9343CrossRefGoogle Scholar
  24. 24.
    Vitrac H, Bogdanov M, Dowhan W (2013) Proper fatty acid composition rather than an ionizable lipid amine is required for full transport function of lactose permease from Escherichia coli. J Biol Chem 288:5873–5885CrossRefGoogle Scholar
  25. 25.
    Tang XB, Casey JR (1999) Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1. Biochemistry 38:14565–14572CrossRefGoogle Scholar
  26. 26.
    Hu YK, Kaplan JH (2000) Site-directed chemical labeling of extracellular loops in a membrane protein. The topology of the Na,K-ATPase alpha-subunit. J Biol Chem 275:19185–19191CrossRefGoogle Scholar
  27. 27.
    Nagamori S, Nishiyama K, Tokuda H (2002) Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA. J Biochem 132:629–634CrossRefGoogle Scholar
  28. 28.
    Dale H, Angevine CM, Krebs MP (2000) Ordered membrane insertion of an archaeal opsin in vivo. Proc Natl Acad Sci U S A 97:7847–7852CrossRefGoogle Scholar
  29. 29.
    Kerr JE, Christie PJ (2010) Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the agrobacterium VirB/VirD4 type IV secretion system. J Bacteriol 192:4923–4934CrossRefGoogle Scholar
  30. 30.
    Xie J, Bogdanov M, Heacock P, Dowhan W (2006) Phosphatidylethanolamine and monoglucosyldiacylglycerol are interchangeable in supporting topogenesis and function of the polytopic membrane protein lactose permease. J Biol Chem 281:19172–19178CrossRefGoogle Scholar
  31. 31.
    Zhang W, Bogdanov M, Pi J, Pittard AJ, Dowhan W (2003) Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition. J Biol Chem 278:50128–50135CrossRefGoogle Scholar
  32. 32.
    Wang X, Bogdanov M, Dowhan W (2002) Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition. EMBO J 21:5673–5681CrossRefGoogle Scholar
  33. 33.
    Bernsel A, Viklund H, Falk J, Lindahl E, von Heijne G, Elofsson A (2008) Prediction of membrane-protein topology from first principles. Proc Natl Acad Sci U S A 105:7177–7181CrossRefGoogle Scholar
  34. 34.
    Zhao G, London E (2006) An amino acid “transmembrane tendency” scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity. Protein Sci 15:1987–2001CrossRefGoogle Scholar
  35. 35.
    Dobson L, Remenyi I, Tusnady GE (2015) CCTOP: a consensus constrained TOPology prediction web server. Nucleic Acids Res 43:W408–W412CrossRefGoogle Scholar
  36. 36.
    Bayer EA, Zalis MG, Wilchek M (1985) 3-(N-Maleimido-propionyl)biocytin: a versatile thiol-specific biotinylating reagent. Anal Biochem 149:529–536CrossRefGoogle Scholar
  37. 37.
    Berezuk AM, Goodyear M, Khursigara CM (2014) Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK. J Biol Chem 289:23287–23301CrossRefGoogle Scholar
  38. 38.
    Moss K, Helm A, Lu Y, Bragin A, Skach WR (1998) Coupled translocation events generate topological heterogeneity at the endoplasmic reticulum membrane. Mol Biol Cell 9:2681–2697CrossRefGoogle Scholar
  39. 39.
    Woodall NB, Yin Y, Bowie JU (2015) Dual-topology insertion of a dual-topology membrane protein. Nat Commun 6:8099CrossRefGoogle Scholar
  40. 40.
    Gafvelin G, von Heijne G (1994) Topological “frustration” in multispanning E. coli inner membrane proteins. Cell 77:401–412CrossRefGoogle Scholar
  41. 41.
    Nasie I, Steiner-Mordoch S, Schuldiner S (2013) Topology determination of untagged membrane proteins. Methods Mol Biol 1033:121–130CrossRefGoogle Scholar
  42. 42.
    Gelis-Jeanvoine S, Lory S, Oberto J, Buddelmeijer N (2015) Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction. Mol Microbiol 95:692–705CrossRefGoogle Scholar
  43. 43.
    Liu Y, Basu A, Li X, Fliegel L (2015) Topological analysis of the Na+/H+ exchanger. Biochim Biophys Acta 1848:2385–2393CrossRefGoogle Scholar
  44. 44.
    Abramson J, Smirnova I, Kasho V, Verner G, Kaback HR, Iwata S (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301:610–615CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biochemistry & Molecular BiologyUniversity of Texas Health Science Center at Houston, McGovern Medical SchoolHoustonUSA

Personalised recommendations