Skip to main content
SpringerLink
Log in

Studying Lipid–Protein Interactions with Electron Paramagnetic Resonance Spectroscopy of Spin-Labeled Lipids

  • Protocol
  • First Online:
Lipid-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 974))

Abstract

Spin label electron paramagnetic resonance (EPR) of lipid–protein interactions reveals crucial features of the structure and assembly of integral membrane proteins. Spin label EPR spectroscopy is the technique of choice to characterize the protein-solvating lipid shell in its highly dynamic nature, because the EPR spectra of lipids that are spin labeled close to the terminal methyl end of their acyl chains display two spectral components, those corresponding to lipids directly contacting the protein and those corresponding to lipids in the bulk fluid bilayer regions of the membrane. In this chapter, typical spin label EPR procedures are presented that allow determination of the stoichiometry of interaction of spin-labeled lipids with the intra-membranous region of membrane proteins or polypeptides, as well as the association constant of the spin-labeled lipid with respect to the host lipid. The lipids giving rise to the so-called immobile spectral component in the EPR spectrum of such samples are identified as the motionally restricted first-shell lipids solvating membrane proteins in biomembranes. Stoichiometry and selectivity are directly related to the structure of the intra-membranous sections of membrane-associated proteins or polypeptides and can be used to study the state of assembly of such proteins in the membrane. Since these characteristics of lipid–protein interactions are discussed in detail in the literature [see Marsh (Eur Biophys J 39:513–525, 2010) for a most recent review], here we focus more on how to spin label model and biomembranes and how to measure and analyze the two-component EPR spectra of spin-labeled lipids in phospholipid bilayers that contain proteins or polypeptides. After a description of how to prepare spin-labeled model and native biological membranes, we present the reader with computational procedures for determining the molar fraction of motionally restricted lipids when both, one, or none of the pure isolated—mobile or immobile—spectral components are available. With these topics, this chapter complements a recent methodological paper [Marsh (Methods 46:83–96, 2008)]. The interpretation of the data is discussed briefly, as well as other relevant and recent spin label EPR techniques for studying lipid–protein interactions, not only from the point of view of lipid chain dynamics.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hidalgo C, Ikemoto N, Gergely J (1976) Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. J Biol Chem 251:4224–4232

    PubMed  CAS  Google Scholar 

  2. Knowles PF, Watts A, Marsh D (1979) Spin-label studies of lipid immobilization in dimyristoylphosphatidylcholine-substituted cytochrome oxidase. Biochemistry 18:4480–4487

    Article  PubMed  CAS  Google Scholar 

  3. Fajer P, Knowles PF, Marsh D (1989) Rotational motion of yeast cytochrome oxidase in phosphatidylcholine complexes studied by saturation-transfer electron spin resonance. Biochemistry 28:5634–5643

    Article  PubMed  CAS  Google Scholar 

  4. Horvath I, Glatz A, Varvasovszki V, Torok Z, Pali T, Balogh G, Kovacs E, Nadasdi L, Benko S, Joo F, Vigh L (1998) Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: Identification of hsp17 as a “fluidity gene”. Proc Natl Acad Sci USA 95:3513–3518

    Article  PubMed  CAS  Google Scholar 

  5. Pali T, Garab G, Horvath LI, Kota Z (2003) Functional significance of the lipid–protein interface in photosynthetic membranes. Cell Mol Life Sci 60:1591–1606

    Article  PubMed  CAS  Google Scholar 

  6. Marsh D (2008) Protein modulation of lipids, and vice-versa, in membranes. Biochim Biophys Acta 1778:1545–1575

    Article  PubMed  CAS  Google Scholar 

  7. Balogh G, Maulucci G, Gombos I, Horvath I, Torok Z, Peter M, Fodor E, Pali T, Benko S, Parasassi T, De Spirito M, Harwood JL, Vigh L (2011) Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells. PLoS One 6:e21182

    Article  PubMed  CAS  Google Scholar 

  8. Berliner LJ (1976) Spin labeling. Theory and applications, molecular biology. An international series of monographs and textbooks 1. Academic Press, New York

    Google Scholar 

  9. Berliner LJ (1979) Spin labeling II. Theory and applications, molecular biology. An international series of monographs and textbooks 2. Academic Press, New York

    Google Scholar 

  10. Berliner LJ, Reuben J (1989) Spin labeling. Theory and applications, biological magnetic resonance 8. Plenum Press, New York and London

    Google Scholar 

  11. Berliner LJ (1998) Spin labeling. The next millennium, biological magnetic resonance 14. Plenum Press, New York and London

    Google Scholar 

  12. Cevc G, Marsh D (1987) Phospholipid bilayers. Physical principles and models, cell biology: a series of monographs 5. John Wiley and Sons, New York

    Google Scholar 

  13. Lasic DD (1993) Liposomes: from physics to applications. Elsevier, Amsterdam

    Google Scholar 

  14. Pali T, Kleinschmidt JH, Powell GL, Marsh D (2000) Nonlinear electron paramagnetic resonance studies of the interaction of cytochrome oxidase with spin-labeled lipids in gel-phase membranes. Biochemistry 39:2355–2361

    Article  PubMed  CAS  Google Scholar 

  15. Jost PC, Griffith OH, Capaldi RA, Vanderkoi G (1973) Evidence for boundary lipid in membranes. Proc Natl Acad Sci USA 70:480–484

    Article  PubMed  CAS  Google Scholar 

  16. Meier P, Sachse J-H, Brophy PJ, Marsh D, Kothe G (1987) Integral membrane proteins significantly decrease the molecular motion in lipid bilayers: a deuteron NMR relaxation study of membranes containing myelin proteolipid apoprotein. Proc Natl Acad Sci USA 84:3704–3708

    Article  PubMed  CAS  Google Scholar 

  17. Marsh D (2008) Electron spin resonance in membrane research: protein–lipid interactions. Methods 46:83–96

    Article  PubMed  CAS  Google Scholar 

  18. Marsh D, Watts A, Pates RD, Uhl R, Knowles PF, Esmann M (1982) ESR spin label studies of lipid–protein interactions in membranes. Biophys J 37:265–274

    Article  PubMed  CAS  Google Scholar 

  19. Marsh D (1983) Spin-label answers to lipid–protein interactions. Trends Biochem Sci 8:330–333

    Article  CAS  Google Scholar 

  20. Marsh D (1987) Selectivity of lipid–protein interactions. J Bioenerg Biomembr 19:677–689

    Article  PubMed  CAS  Google Scholar 

  21. Marsh D (1990) Lipid–protein interactions in membranes. FEBS Lett 268:371–375

    Article  PubMed  CAS  Google Scholar 

  22. Knowles PF, Marsh D (1991) Magnetic resonance of membranes. Biochem J 274:625–641

    PubMed  CAS  Google Scholar 

  23. Marsh D, Horvath LI (1998) Structure, dynamics and composition of the lipid–protein interface. Perspectives from spin-labelling. Biochim Biophys Acta 1376:267–296

    Article  PubMed  CAS  Google Scholar 

  24. Kota Z, Pali T, Marsh D (2004) Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance. Biophys J 86:1521–1531

    Article  PubMed  CAS  Google Scholar 

  25. Marsh D, Pali T (2004) The protein–lipid interface: perspectives from magnetic resonance and crystal structures. Biochim Biophys Acta 1666:118–141

    Article  PubMed  CAS  Google Scholar 

  26. Pali T, Bashtovyy D, Marsh D (2006) Stoichiometry of lipid interactions with transmembrane proteins – deduced from the 3D structures. Protein Sci 15:1153–1161

    Article  PubMed  CAS  Google Scholar 

  27. Griffith OH, Jost PC (1976) Lipid spin labels in biological membranes. In: Berliner LJ (ed) Spin labeling. Theory and applications. Academic Press, New York, pp 453–523

    Google Scholar 

  28. McConnell HM (1976) Molecular motion in biological membranes. In: Berliner LJ (ed) Spin labeling. Theory and applications. Academic Press, New York, pp 525–560

    Google Scholar 

  29. Seelig J (1976) Anisotropic motion in liquid crystalline structures. In: Berliner LJ (ed) Spin labeling. Theory and applications. Academic Press, New York, pp 373–409

    Google Scholar 

  30. Lange A, Marsh D, Wassmer KH, Meier P, Kothe G (1985) Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model. Biochemistry 24:4383–4392

    Article  PubMed  CAS  Google Scholar 

  31. Marsh D (1989) Experimental methods in spin-label spectral analysis. In: Berliner LJ, Reuben J (eds) Spin labeling. Theory and applications. Plenum Press, New York and London, pp 255–303

    Chapter  Google Scholar 

  32. Moser M, Marsh D, Meier P, Wassmer K-H, Kothe G (1989) Chain configuration and flexibility gradient in phospholipid membranes. Comparison between spin-label electron spin resonance and deuteron nuclear magnetic resonance, and identification of new conformations. Biophys J 55:111–123

    Article  PubMed  CAS  Google Scholar 

  33. Horvath LI, Brophy PJ, Marsh D (1994) Microwave frequency dependence of ESR spectra from spin labels undergoing two-site exchange in myelin proteolipid membranes. J Magn Reson B 105:120–128

    Article  PubMed  CAS  Google Scholar 

  34. Marsh D, Kurad D, Livshits VA (2002) High-field electron spin resonance of spin labels in membranes. Chem Phys Lipids 116:93–114-PII S0009-3084(02)00022-1

    Google Scholar 

  35. Marsh D, Pali T, Horvath LI (1998) Progressive saturation and saturation transfer EPR for measuring exchange processes and proximity relations in membranes. In: Berliner LJ (ed) Spin labeling, the next millennium. Plenum Press, New York and London, pp 23–82

    Google Scholar 

  36. Pali T, Finbow ME, Marsh D (1999) Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR. Biochemistry 38:14311–14319

    Article  PubMed  CAS  Google Scholar 

  37. Marsh D (2010) Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art. Eur Biophys J 39:513–525

    Article  PubMed  CAS  Google Scholar 

  38. Esmann M, Hideg K, Marsh D (1988) Novel spin-labels for the study of lipid–protein interactions. Application to (Na+, K+)-ATPase membranes. Biochemistry 27:3913–3917

    Article  PubMed  CAS  Google Scholar 

  39. Horvath LI, Brophy PJ, Marsh D (1993) Exchange rates at the lipid–protein interface of the myelin proteolipid protein determined by saturation transfer electron spin resonance and continuous wave saturation studies. Biophys J 64:622–631

    Article  PubMed  CAS  Google Scholar 

  40. Horvath LI, Brophy PJ, Marsh D (1993) Spin label saturation transfer EPR determinations of the stoichiometry and selectivity of lipid–protein interactions in the gel phase. Biochim Biophys Acta 1147:277–280

    Article  PubMed  CAS  Google Scholar 

  41. Marsh D, Pali T (2006) Lipid conformation in crystalline bilayers and in crystals of transmembrane proteins. Chem Phys Lipids 141:48–65

    Article  PubMed  CAS  Google Scholar 

  42. Horvath LI, Arias HR, Hankovszky HO, Hideg K, Barrantes FJ, Marsh D (1990) Association of spin-labeled local anaesthetics at the hydrophobic surface of acetylcholine receptor in native membranes from Torpedo marmorata. Biochemistry 29:8707–8713

    Article  PubMed  CAS  Google Scholar 

  43. Dixon N, Pali T, Kee TP, Marsh D (2004) Spin-labelled vacuolar-ATPase inhibitors in lipid membranes. Biochim Biophys Acta Biomembr 1665:177–183

    Article  CAS  Google Scholar 

  44. Dixon N, Pali T, Kee TP, Ball S, Harrison MA, Findlay JBC, Nyman J, Vaananen K, Finbow ME, Marsh D (2008) Interaction of spin-labeled inhibitors of the vacuolar H+−ATPase with the transmembrane Vo-sector. Biophys J 94:506–514

    Article  PubMed  CAS  Google Scholar 

  45. Pali T, Horvath LI (1989) Restricted lateral diffusion of acidic lipids in phospholipid vesicles aggregated by myelin basic protein. Biochim Biophys Acta 984:128–134

    Article  CAS  Google Scholar 

  46. Stopar D, Jansen KAJ, Pali T, Marsh D, Hemminga MA (1997) Membrane location of spin-labeled M13 major coat protein mutants determined by paramagnetic relaxation agents. Biochemistry 36:8261–8268

    Article  PubMed  CAS  Google Scholar 

  47. Marsh D, Livshits VA, Pali T, Gaffney BJ (1999) Recent development in biological spin-label spectroscopy. In: Greve J, Puppels GJ, Otto C (eds) Spectroscopy of biological molecules: new directions. Kluwer Academic Publishers, Dordrecht, Boston, London, pp 647–650

    Chapter  Google Scholar 

  48. Kostrzewa A, Pali T, Froncisz W, Marsh D (2000) Membrane location of spin-labeled cytochrome determined by paramagnetic relaxation agents. Biochemistry 39:6066–6074

    Article  PubMed  CAS  Google Scholar 

  49. Kota Z, Szalontai B, Droppa M, Horvath G, Pali T (2002) The formation of an inverted hexagonal phase from thylakoid membranes upon heating. Cell Mol Biol Lett 7:126–128

    PubMed  CAS  Google Scholar 

  50. Pali T, Marsh D (2002) Structural studies on membrane proteins using non-linear spin label EPR spectroscopy. Cell Mol Biol Lett 7:87–91

    PubMed  CAS  Google Scholar 

  51. Livshits VA, Marsh D (2000) Fatty acid binding sites of serum albumin probed by non-linear spin-label EPR. Biochim Biophys Acta 1466:350–360

    Article  PubMed  CAS  Google Scholar 

  52. De Simone F, Guzzi R, Sportelli L, Marsh D, Bartucci R (2007) Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin. Biochim Biophys Acta 1768:1541–1549

    Article  PubMed  CAS  Google Scholar 

  53. Brotherus JR, Griffith OH, Brotherus MO, Jost PC, Silvius JR, Hokin LE (1981) Lipid– protein multiple binding equilibriums in membranes. Biochemistry 20:5261–5267

    Article  PubMed  CAS  Google Scholar 

  54. McConnell HM, Hubbell WL (1971) Molecular motion in spin-labeled phospholipids and membranes. J Am Chem Soc 93:314–326

    Article  PubMed  CAS  Google Scholar 

  55. Marsh D, Watts A (1982) [90] Diffusible spin labels used to study lipid–protein interactions with rhodopsin and bacteriorhodopsin. Methods Enzymol 88:762–772

    Article  CAS  Google Scholar 

  56. Esmann M, Marsh D (1985) Spin-label studies on the origin of the specificity of lipid–protein interactions in Na+, K+−ATPase membranes from Squalus acanthias. Biochemistry 24:3572–3578

    Article  PubMed  CAS  Google Scholar 

  57. Sankaram MB, Brophy PJ, Jordi W, Marsh D (1990) Fatty acid pH titration and the selectivity of interaction with extrinsic proteins in dimyristoylphosphatidylglycerol dispersions. Spin label ESR studies. Biochim Biophys Acta 1021:63–69

    Article  CAS  Google Scholar 

  58. Pali T, Finbow ME, Holzenburg A, Findlay JBC, Marsh D (1995) Lipid–protein interactions and assembly of the 16-kDa channel polypeptide from Nephrops norvegicus. Studies with spin-label electron spin resonance spectroscopy and electron microscopy. Biochemistry 34:9211–9218

    Article  PubMed  CAS  Google Scholar 

  59. Kota Z, Horvath LI, Droppa M, Horvath G, Farkas T, Pali T (2002) Protein assembly and heat stability in developing thylakoid membranes during greening. Proc Natl Acad Sci USA 99:12149–12154

    Article  PubMed  CAS  Google Scholar 

  60. Kleinschmidt JH, Marsh D (1997) Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes. Biophys J 73:2546–2555

    Article  PubMed  CAS  Google Scholar 

  61. Kleinschmidt JH, Powell GL, Marsh D (1998) Cytochrome c-induced increase of motionally restricted lipid in reconstituted cytochrome c oxidase membranes, revealed by spin-label ESR spectroscopy. Biochemistry 37:11579–11585

    Article  PubMed  CAS  Google Scholar 

  62. Kiricsi M, Horvath LI, Dux L, Pali T (2001) Spin label EPR studies of the effect of gramicidin S on lipid chain dynamics. J Mol Struct 563:469–475

    Article  Google Scholar 

  63. Marsh D, Horvath LI, Swamy MJ, Mantripragada S, Kleinschmidt JH (2002) Interaction of membrane-spanning proteins with peripheral and lipid-anchored membrane proteins: perspectives from protein–lipid interactions. Mol Membr Biol 19:247–255

    Article  PubMed  CAS  Google Scholar 

  64. Anbazhagan V, Qu J, Kleinschmidt JH, Marsh D (2008) Incorporation of outer membrane protein OmpG in lipid membranes: protein– lipid interactions and beta-barrel orientation. Biochemistry 47:6189–6198

    Article  PubMed  CAS  Google Scholar 

  65. Kota Z, Pali T, Dixon N, Kee TP, Harrison MA, Findlay JB, Finbow ME, Marsh D (2008) Incorporation of transmembrane peptides from the vacuolar H(+)-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy. Biochemistry 47:3937–3949

    Article  PubMed  CAS  Google Scholar 

  66. Watts A, Davoust J, Marsh D, Devaux PF (1981) Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results. Biochim Biophys Acta 643:673–676

    Article  PubMed  CAS  Google Scholar 

  67. Ivancich A, Horvath LI, Droppa M, Horvath G, Farkas T (1994) Spin label ESR study of lipid solvation of supramolecular photosynthetic protein complexes in thylakoids. Biochim Biophys Acta Biomembr 1196:51–56

    Article  CAS  Google Scholar 

  68. Eibl H, Lands WE (1969) A new, sensitive determination of phosphate. Anal Biochem 30:51–57

    Article  PubMed  CAS  Google Scholar 

  69. Rouser G, Fleischer S, Yamamoto A (1970) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorous analysis of spots. Lipids 5:494–496

    Article  PubMed  CAS  Google Scholar 

  70. Lowry OH, Rosebrough NJ, Farr L, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  71. Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356

    Article  PubMed  CAS  Google Scholar 

  72. Markwell MAK, Haas SM, Tolbert NE, Bieber LL (1981) Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol 72:296–303

    Article  PubMed  CAS  Google Scholar 

  73. Gasser A, Raddatz S, Radunz A, Schmid G (1999) Comparative immunological and chemical analysis of lipids and carotenoids of the D1-peptide and of the light-harvesting- complex of photosystem II of Nicotiana tabacum. Zeitsch Naturforsch C J Biosci 54:199–208

    CAS  Google Scholar 

  74. Kruse O, Hankamer B, Konczak C, Gerle C, Morris E, Radunz A, Schmid G, Barber J (2000) Phosphatidylglycerol is involved in the dimerization of photosystem II. J Biol Chem 275:6509–6514

    Article  PubMed  CAS  Google Scholar 

  75. Mongrand S, Badoc A, Patouille B, Lacomblez C, Chavent M, Cassagne C, Bessoule J (2001) Taxonomy of gymnospermae: multivariate analyses of leaf fatty acid composition. Phytochemistry 58:101–115

    Article  PubMed  CAS  Google Scholar 

  76. Perez-Gil J, Casals C, Marsh D (1995) Interactions of hydrophobic lung surfactant proteins SP-B and SP-C with dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol bilayers studied by electron spin resonance spectroscopy. Biochemistry 34:3964–3971

    Article  PubMed  CAS  Google Scholar 

  77. Cruz A, Casals C, Plasencia I, Marsh D, Perez-Gil J (1998) Depth profiles of pulmonary surfactant protein B in phosphatidylcholine bilayers, studied by fluorescence and electron spin resonance. Biochemistry 37:9488–9496

    Article  PubMed  CAS  Google Scholar 

  78. Peelen SJCJ, Sanders JC, Hemminga MA, Marsh D (1992) Stoichiometry, selectivity, and exchange dynamics of lipid–protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure. Biochemistry 31:2670–2677

    Article  PubMed  CAS  Google Scholar 

  79. Aggeli A, Boden N, Cheng Y-L, Findlay JBC, Knowles PF, Kovatchev P, Turnbull JHP (1996) Peptides modeled on the transmembrane region of the slow voltage-gated IsK potassium channel: structural characterization of peptide assemblies in the beta-strand conformation. Biochemistry 35:16213–16221

    Article  PubMed  CAS  Google Scholar 

  80. Kleinschmidt JH, Tamm LK (1996) Folding intermediates of a beta-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism. Biochemistry 35:12993–13000

    Article  PubMed  CAS  Google Scholar 

  81. Wolkers WF, Spruijt RB, Kaan A, Konings RNH, Hemminga MA (1997) Conventional and saturation-transfer EPR of spin-labeled mutant bacteriophage M13 coat protein in phospholipid bilayers. Biochim Biophys Acta 1327:5–16

    Article  PubMed  CAS  Google Scholar 

  82. Kleinschmidt JH, den Blaauwen T, Driessen AJ, Tamm LK (1999) Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism. Biochemistry 38:5006–5016

    Article  PubMed  CAS  Google Scholar 

  83. Bosterling B, Stier A, Hildebrandt AG, Dawson JH, Trudell JR (1979) Reconstitution of cytochrome P-450 and cytochrome P-450 reductase into phosphatidylcholine-phosphatidylethanolamine bilayers: characterization of structure and metabolic activity. Mol Pharmacol 16:332–342

    PubMed  CAS  Google Scholar 

  84. Powell GL, Knowles PF, Marsh D (1987) Spin label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase. Biochemistry 26:8138–8145

    Article  PubMed  CAS  Google Scholar 

  85. Horvath LI, Heimburg T, Kovachev P, Findlay JBC, Hideg K, Marsh D (1995) Integration of a K+ channel-associated peptide in a lipid bilayer: conformation. Lipid–protein interactions, and rotational diffusion. Biochemistry 34:3893–3898

    Article  PubMed  CAS  Google Scholar 

  86. Yang Q, Liu X, Hara M, Lundahl P, Miyake J (2000) Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Biochem 280:94–102

    Article  PubMed  CAS  Google Scholar 

  87. Yu C, Yu L, King TE (1975) Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c. J Biol Chem 250:1383–1392

    PubMed  CAS  Google Scholar 

  88. Cable MB, Powell GL (1980) Spin-labeled cardiolipin: preferential segregation in the boundary layer of cytochrome c oxidase. Biochemistry 19:5679–5686

    Article  PubMed  CAS  Google Scholar 

  89. Watts A, Marsh D, Knowles PF (1978) Lipid-substituted cytochrome oxidase: no absolute requirement of cardiolipin for activity. Biochem Biophys Res Commun 81:403–409

    Article  PubMed  CAS  Google Scholar 

  90. Yonetani T (1966) Cytochrome oxidase from beef heart muscle. In: Maehly AC (ed) Biochemical preparations, vol 11. John Wiley & Sons, New York, pp 14–20

    Google Scholar 

  91. Gombos Z, Kis M, Pali T, Vigh L (1987) Nitrate starvation induces homeoviscous regulation of lipids in the cell envelope of the blue-green alga, Anacystis nidulans. Eur J Biochem 165:461–465

    Article  PubMed  CAS  Google Scholar 

  92. Horvath I, Vigh L, Pali T, Thompson GA Jr (1989) Effect of catalytic hydrogenation of Tetrahymena ciliary phospholipid fatty acids on ciliary phospholipase A activity. Biochim Biophys Acta 1002:409–412

    Article  PubMed  CAS  Google Scholar 

  93. Pali T, Pesti M (1996) Chapter V: phase transition of membrane lipids. In: Prasad R (ed) Manual on membrane lipids. Springer-Verlag, Berlin, Heidelberg, New York, pp 80–93

    Google Scholar 

  94. Pali T, Finbow ME, Marsh D (1997) Membrane assembly of the 16-kDa V-ATPase proteolipid subunit from spin–lattice relaxation enhancements in spin label ESR. Biophys J 72:TUAM7

    Google Scholar 

  95. Belagyi J, Pas M, Rasport P, Pesti M, Pali T (1999) Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy. Biochim Biophys Acta Biomembr 1421:175–182

    Article  CAS  Google Scholar 

  96. Finbow ME, Eldridge T, Buultjens J, Lane NJ, Shuttleworth J, Pitts JD (1984) Isolation and characterisation of arthropod gap-junctions. EMBO J 3:2271–2278

    PubMed  CAS  Google Scholar 

  97. Hertzberg EL (1984) A detergent-independent procedure for the isolation of gap-junctions from rat-liver. J Biol Chem 259:9936–9943

    PubMed  CAS  Google Scholar 

  98. Buultjens TEJ, Finbow ME, Lane NJ, Pitts JD (1988) Tissue and species conservation of the vertebrate and arthropod forms of the low-molecular weight (16–18000) proteins of gap-junctions. Cell Tissue Res 251:571–580

    Article  PubMed  CAS  Google Scholar 

  99. Leitch B, Finbow ME (1990) The gap junction-like form of a vacuolar proton channel component appears not to be an artifact of isolation – an immunocytochemical localization study. Exp Cell Res 190:218–226

    Article  PubMed  CAS  Google Scholar 

  100. Finbow ME, Eliopoulos EE, Jackson PJ, Keen JN, Meagher L, Thomson P, Jones P, Findlay JBC (1992) Structure of a 16 kDa integral membrane protein that has identity to the putative proton channel of the vacuolar H+−ATPase. Protein Eng 5:7–15

    Article  PubMed  CAS  Google Scholar 

  101. Finbow ME, Meagher L (1992) Connexins and the vacuolar proteolipid-like 16-kDa protein are not directly associated with each other but may be components of similar or the same gap junctional complexes. Exp Cell Res 203:280–284

    Article  PubMed  CAS  Google Scholar 

  102. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  PubMed  CAS  Google Scholar 

  103. Folch JM, Lees M, Sloane SGH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509

    PubMed  CAS  Google Scholar 

  104. Horvath G, Droppa M, Hideg E, Rozsa Z (1989) The role of phospholipids in regulating photosynthetic electron transport activities: treatment of chloroplasts with phospholipase A2. J Photochem Photobiol B Biol 3:515–527

    Article  CAS  Google Scholar 

  105. Jost PC, Griffith OH (1976) Instrumental aspects of spin labeling. In: Berliner LJ (ed) Spin labeling. Theory and applications. Academic Press, New York, pp 251–272

    Google Scholar 

  106. Datema KP, Wolfs CJAM, Marsh D, Watts A, Hemminga MA (1987) Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers. Biochemistry 26:7571–7574

    Article  PubMed  CAS  Google Scholar 

  107. Ryba NJP, Horvath LI, Watts A, Marsh D (1987) Molecular exchange at the lipid-rhodopsin interface: spin-label electron spin resonance studies of rhodopsin-dimyristoyl phosphatidylcholine recombinants. Biochemistry 26:3234–3240

    Article  PubMed  CAS  Google Scholar 

  108. Horvath LI, Brophy PJ, Marsh D (1988) Influence of lipid headgroup on the specificity and exchange dynamics in lipid–protein interactions. A spin label study of myelin proteolipid apoprotein-phospholipid complexes. Biochemistry 27:5296–5304

    Article  PubMed  CAS  Google Scholar 

  109. Li G, Knowles PF, Murphy DJ, Nishida I, Marsh D (1989) Spin-label ESR studies of lipid–protein interactions in thylakoid membranes. Biochemistry 28:7446–7452

    Article  PubMed  CAS  Google Scholar 

  110. Li G, Knowles PF, Murphy DJ, Marsh D (1990) Lipid–protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy. J Biol Chem 265:16867–16872

    PubMed  CAS  Google Scholar 

  111. Marsh D (1996) Membrane assembly studied by spin-label electron spin resonance. Braz J Med Biol Res 29:863–871

    PubMed  CAS  Google Scholar 

  112. Mihailescu D, Horvath LI (1999) Molecular dynamics of lipid association at the hydrophobic interface of gramicidin S. Eur Biophys J 28:216–221

    Article  CAS  Google Scholar 

  113. Szakonyi G, Dux L, Horvath LI (1999) Rearrangement of boundary, stand-by, and fluid lipids during the formation of two-dimensional crystals of Ca2+ −ATPase. J Mol Struct 483:253–256

    Article  Google Scholar 

  114. Marsh D (1982) Electron spin resonance: spin label probes. In: Metcalfe JC, Hesketh TR (eds) Techniques in lipid and membrane biochemistry. Elsevier, Amsterdam, p B426/1–B426/44

    Google Scholar 

  115. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55

    Article  PubMed  CAS  Google Scholar 

  116. Brophy PJ, Horvath LI, Marsh D (1984) Stoichiometry and specificity of lipid–protein Interaction with myelin proteolipid protein studied by spin-label electron spin resonance. Biochemistry 23:860–865

    Article  PubMed  CAS  Google Scholar 

  117. Marsh D (1985) ESR spin label studies of lipid–protein interactions, vol 1, Chapter 4. In: Watts A, Pont JJHHM (eds) Progress in protein–lipid interactions. Elsevier, Amsterdam, pp 143–172

    Google Scholar 

  118. Marsh D, Horvath LI (1989) Spin-label studies of the structure dynamics, of lipids proteins. In: Hoff AJ (ed) Advanced EPR. Applications in biology and biochemistry. Elsevier, Amsterdam, pp 707–752

    Google Scholar 

  119. Horvath LI, Drees M, Beyer K, Klingenberg M, Marsh D (1990) Lipid–protein Interactions in ADP-ATP carrier/egg phosphatidylcholine recombinants studied by spin-label ESR spectroscopy. Biochemistry 29:10664–10669

    Article  PubMed  CAS  Google Scholar 

  120. Goerrissen H, Marsh D, Rietveld A, de Kruijff B (1986) Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance. Biochemistry 25:2904–2910

    Article  CAS  Google Scholar 

  121. Jordi W, de Kruijff B, Marsh D (1989) Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study. Biochemistry 28:8998–9005

    Article  PubMed  CAS  Google Scholar 

  122. Montich GG, Montecucco C, Papini E, Marsh D (1995) Insertion of diphtheria toxin in lipid bilayers studied by spin label ESR. Biochemistry 34:11561–11567

    Article  PubMed  CAS  Google Scholar 

  123. Horvath LI, Brophy PJ, Marsh D (1990) Influence of polar residue deletions on lipid–protein interactions with the myelin proteolipid protein. Spin-label ESR studies with DM-20/lipid recombinants. Biochemistry 29:2635–2638

    Article  PubMed  CAS  Google Scholar 

  124. Freed JH (1976) Theory of slow tumbling ESR spectra of nitroxides. In: Berliner LJ (ed) Spin labeling. Theory and applications. Academic Press, New York, pp 53–132

    Google Scholar 

  125. Marsh D, Watts A, Maschke W, Knowles PF (1978) Protein-immobilized lipid in dimyristoylphosphatidylcholine-substituted cytochrome oxidase: evidence for both boundary and trapped-bilayer lipid. Biochem Biophys Res Commun 81:397–402

    Article  PubMed  CAS  Google Scholar 

  126. Arsov Z, Schara M, Zorko M, Strancar J (2004) The membrane lateral domain approach in the studies of lipid–protein interaction of GPI-anchored bovine erythrocyte acetylcholinesterase. Eur Biophys J 33:715–725

    Article  PubMed  CAS  Google Scholar 

  127. Arsov Z, Strancar J (2005) Determination of partition coefficient of spin probe between different lipid membrane phases. J Chem Inf Model 45:1662–1671

    Article  PubMed  CAS  Google Scholar 

  128. Strancar J, Schara M, Pecar S (2003) New EPR method for cellular surface characterization. J Membr Biol 193:15–22

    Article  PubMed  CAS  Google Scholar 

  129. Pali T, Horvath LI (1989) Lipid lateral diffusion measurements in model membranes. Acta Physiol Hung 74:311–314

    PubMed  CAS  Google Scholar 

  130. Davoust J, Devaux PF (1982) Simulation of electron spin resonance spectra of spin-labeled fatty acids covalently attached to the boundary of an intrinsic membrane protein. A chemical exchange model. J Magn Reson 48:475–494

    CAS  Google Scholar 

  131. Horvath LI, Brophy PJ, Marsh D (1988) Exchange rates at the lipid–protein interface of myelin proteolipid protein studied by spin-label electron spin resonance. Biochemistry 27:46–52

    Article  PubMed  CAS  Google Scholar 

  132. Wolfs CJAM, Horvath LI, Marsh D, Watts A, Hemminga MA (1989) Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage coat protein in lipid bilayers. Biochemistry 28:9995–10001

    Article  PubMed  CAS  Google Scholar 

  133. Ryba N, Horvath LI, Watts A, Marsh D (1987) Exchange at the lipid–protein interface. Spin label ESR studies of rhodopsin-dimyristoyl phosphatidylcholine recombinants. Biochemistry 26:3234–3240

    Article  PubMed  CAS  Google Scholar 

  134. Sankaram MB, de Kruijff B, Marsh D (1989) Selectivity of interaction of spin-labelled lipids with peripheral proteins bound to dimyristoylphosphatidylglycerol bilayers, as determined by ESR spectroscopy. Biochim Biophys Acta 986:315–320

    Article  CAS  Google Scholar 

  135. Sankaram MB, Brophy PJ, Marsh D (1989) Interaction of two complementary fragments of the bovine spinal cord myelin basic protein with phospholipid bilayers. An ESR spin label study. Biochemistry 28:9692–9698

    Article  PubMed  CAS  Google Scholar 

  136. Pali T, Tigyi G, Horvath LI (1988) Constrained lateral diffusion of membrane lipids as a consequence of myelin basic protein/lipid interaction. Acta Physiol Hung 71:146

    Google Scholar 

  137. Pali T, Ebert B, Horvath LI (1987) ESR imaging of myelin basic protein induced vesicle aggregation. Biochim Biophys Acta Biomembr 904:346–352

    Article  CAS  Google Scholar 

  138. Pali T, Sass L, Ebert B, Horvath LI (1990) 2D ESR image reconstruction from 1D projections using the modulated field gradient method. J Magn Reson 86:338–345

    CAS  Google Scholar 

  139. Pali T, Ebert B, Horvath LI (1992) Dynamic imaging and spatially localized ESR spectroscopy of oriented phospholipid multilayers. J Magn Reson 96:491–500

    CAS  Google Scholar 

  140. Marsh D, Livshits VA, Pali T (1997) Non-linear, continuous-wave EPR spectroscopy and spin–lattice relaxation: spin-label EPR methods for structure and dynamics. J Chem Soc Perkin Trans 2:2545–2548

    Google Scholar 

  141. Pali T, Bartucci R, Horvath LI, Marsh D (1993) Kinetics and dynamics of annealing during sub-gel phase formation in phospholipid bilayers. A saturation transfer electron spin resonance study. Biophys J 64:1781–1788

    Article  PubMed  CAS  Google Scholar 

  142. Pali T, Bartucci R, Horvath LI, Marsh D (1992) Distance measurements using paramagnetic ion-induced relaxation in the saturation transfer electron spin resonance of spin-labeled biomolecules. Biophys J 61:1595–1602

    Article  PubMed  CAS  Google Scholar 

  143. Pali T, Finbow ME, Marsh D (2006) A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus-revealed by EPR spectroscopy. Biochim Biophys Acta Biomembr 1758:206–212

    Article  CAS  Google Scholar 

  144. Arora A, Esmann M, Marsh D (1999) Microsecond motions of the lipids associated with trypsinized Na, K-ATPase membranes. Progressive saturation spin-label electron spin resonance studies. Biochemistry 38:10084–10091

    Article  PubMed  CAS  Google Scholar 

  145. Gaffney BJ, Marsh D (1998) High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes. Proc Natl Acad Sci USA 95:12940–12943

    Article  PubMed  CAS  Google Scholar 

  146. Livshits VA, Marsh D (2000) Simulation studies of high-field EPR spectra of spin-labeled lipids in membranes. J Magn Reson 147:59–67

    Article  PubMed  CAS  Google Scholar 

  147. Bartucci R, Gambacorta A, Gliozzi A, Marsh D, Sportelli L (2005) Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance. Biochemistry 44:15017–15023

    Article  PubMed  CAS  Google Scholar 

  148. Bartucci R, Erilov DA, Guzzi R, Sportelli L, Dzuba SA, Marsh D (2006) Time-resolved electron spin resonance studies of spin-labelled lipids in membranes. Chem Phys Lipids 141:142–157

    Article  PubMed  CAS  Google Scholar 

  149. Hilger D, Polyhach Y, Padan E, Jung H, Jeschke G (2007) High-resolution structure of a Na+/H+ antiporter dimer obtained by pulsed electron paramagnetic resonance distance measurements. Biophys J 93:3675–3683

    Article  PubMed  CAS  Google Scholar 

  150. Volkov A, Dockter C, Bund T, Paulsen H, Jeschke G (2009) Pulsed EPR determination of water accessibility to spin-labeled amino acid residues in LHCIIb. Biophys J 96:1124–1141

    Article  PubMed  CAS  Google Scholar 

  151. Dockter C, Volkov A, Bauer C, Polyhach Y, Joly-Lopez Z, Jeschke G, Paulsen H (2009) Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR. Proc Nat Acad Sci USA 106:18485–18490

    Article  PubMed  CAS  Google Scholar 

  152. Dockter C, Mueller AH, Dietz C, Volkov A, Polyhach Y, Jeschke G, Paulsen H (2011) Rigid core and flexible terminus. Structure of solubilized light-harvesting chlorophyll a/b complex (LHCII) measured by electron paramagnetic resonance (EPR). J Biol Chem 287:2915–2926

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

The library spectra used in this chapter for illustrations were recorded, and the optimized subtraction routines were started to be developed (by T.P.) in Derek Marsh’s laboratory in Göttingen, Germany. We thank him also for reading the manuscript. We acknowledge financial support from the Hungarian National Science Fund (OTKA K68804 and K101633).

Author information

Authors and Affiliations

  1. Institute of Biophysics, Biological Research Centre, Szeged, Hungary

    Tibor Páli & Zoltán Kóta

Authors
  1. Tibor Páli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zoltán Kóta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tibor Páli .

Editor information

Editors and Affiliations

  1. Institut für Biologie, Abteilung Biophysik, Universität Kassel, Heinrich-Plett-Straße 40, Kassel, 34132, Germany

    Jörg H. Kleinschmidt

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Páli, T., Kóta, Z. (2013). Studying Lipid–Protein Interactions with Electron Paramagnetic Resonance Spectroscopy of Spin-Labeled Lipids. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 974. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-275-9_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-275-9_14

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-274-2

  • Online ISBN: 978-1-62703-275-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation