Solid-State 19F-NMR Analysis of Peptides in Oriented Biomembranes

  • Erik StrandbergEmail author
  • Anne S. Ulrich
Reference work entry


Solid-state 19F-NMR is a powerful method used to study membrane-active peptides under quasi-native conditions in lipid bilayers. As shown in this chapter, it is possible to determine the conformation, orientation, dynamics, and aggregation behavior of peptides in membranes. Compared with other nuclei, 19F provides a stronger signal, thus making it possible to study peptides at low concentrations (at peptide-to-lipid molar ratios as low as 1:3000) and to determine long distances between two labels (up to 11 Å in fluid bilayers). The method is well established and has been applied to a number of antimicrobial, cell-penetrating, and fusogenic peptides that form α-helices, β-sheets, or more irregular structures, and the results are summarized here. Several new 19F-labeled amino acids that have been recently introduced are described. With these, it is now possible to replace not only hydrophobic but also polar or charged amino acids without significant perturbation, thus making solid-state 19F-NMR a highly versatile tool for characterizing peptide-lipid interactions.


Alpha-helical peptides Antimicrobial peptides Beta-sheet peptides Cell-penetrating peptides Distance measurements Fusogenic peptides Membrane-active peptides Oriented samples Peptide orientation, dynamics and aggregation Solid-state 19F-NMR 19F-labeled amino acids 19F-19F dipolar couplings 


  1. 1.
    Wadhwani P, Tremouilhac P, Strandberg E, Afonin S, Grage SL, Ieronimo M, Berditsch M, Ulrich AS. Using fluorinated amino acids for structure analysis of membrane-active peptides by solid-state 19F-NMR. In: Soloshonok VA, Mikami K, Yamazaki T, Welch JT, Honek JF, editors. Current fluoroorganic chemistry: new synthetic directions, technologies, materials, and biological applications. Washington, DC: American Chemical Society; 2007. p. 431–46.CrossRefGoogle Scholar
  2. 2.
    Ulrich AS, Wadhwani P, Dürr UHN, Afonin S, Glaser RW, Strandberg E, Tremouilhac P, Sachse C, Berditchevskaia M, Grage SL. Solid-state 19F-nuclear magnetic resonance analysis of membrane-active peptides. In: Ramamoorthy A, editor. NMR spectroscopy of biological solids. Boca Raton: CRC Press; 2006. p. 215–36.Google Scholar
  3. 3.
    Ulrich AS. Solid state 19F-NMR methods for studying biomembranes. Prog Nucl Magn Reson Spectrosc. 2005;46:1–21.CrossRefGoogle Scholar
  4. 4.
    Koch K, Afonin S, Ieronimo M, Berditsch M, Ulrich AS. Solid-state 19F-NMR of peptides in native membranes. Top Curr Chem. 2012;306:89–118.CrossRefGoogle Scholar
  5. 5.
    Ulrich AS. Solid state 19F-NMR analysis of oriented biomembranes. In: Webb GA, editor. Modern magnetic resonance. Dordrecht: Springer; 2007. p. 261–7.Google Scholar
  6. 6.
    Ieronimo M, Afonin S, Koch K, Berditsch M, Wadhwani P, Ulrich AS. 19F NMR analysis of the antimicrobial peptide PGLa bound to native cell membranes from bacterial protoplasts and human erythrocytes. J Am Chem Soc. 2010;132:8822–4.CrossRefGoogle Scholar
  7. 7.
    Misiewicz J, Afonin S, Grage SL, van den Berg J, Strandberg E, Wadhwani P, Ulrich AS. Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR. J Biomol NMR. 2015;61:287–98.CrossRefGoogle Scholar
  8. 8.
    Glaser RW, Ulrich AS. Susceptibility corrections in solid-state NMR experiments with oriented membrane samples. Part I: applications. J Magn Reson. 2003;164:104–14.CrossRefGoogle Scholar
  9. 9.
    Wadhwani P, Strandberg E, Heidenreich N, Bürck J, Fanghänel S, Ulrich AS. Self-assembly of flexible β-strands into immobile amyloid-like β-sheets in membranes as revealed by solid-state 19F NMR. J Am Chem Soc. 2012;134:6512–5.CrossRefGoogle Scholar
  10. 10.
    Gilchrist Jr ML, Monde K, Tomita Y, Iwashita T, Nakanishi K, McDermott AE. Measurement of interfluorine distances in solids. J Magn Reson. 2001;152:1–6.CrossRefGoogle Scholar
  11. 11.
    Salgado J, Grage SL, Kondejewski LH, Hodges RS, McElhaney RN, Ulrich AS. Membrane-bound structure and alignment of the antimicrobial β-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR. J Biomol NMR. 2001;21:191–208.CrossRefGoogle Scholar
  12. 12.
    Grage SL, Xu X, Schmitt M, Wadhwani P, Ulrich AS. 19F-labeling of peptides revealing long-range NMR distances in fluid membranes. J Phys Chem Lett. 2014;5:4256–9.CrossRefGoogle Scholar
  13. 13.
    Grage SL, Suleymanova AV, Afonin S, Wadhwani P, Ulrich AS. Solid state NMR analysis of the dipolar couplings within and between distant CF3-groups in a membrane-bound peptide. J Magn Reson. 2006;183:77–86.CrossRefGoogle Scholar
  14. 14.
    Ulrich R, Glaser RW, Ulrich AS. Susceptibility corrections in solid state NMR experiments with macroscopically oriented membrane samples. Part II: theory. J Magn Reson. 2003;164:115–27.CrossRefGoogle Scholar
  15. 15.
    Glaser RW, Sachse C, Dürr UHN, Wadhwani P, Ulrich AS. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels. J Magn Reson. 2004;168:153–63.CrossRefGoogle Scholar
  16. 16.
    Mikhailiuk PK, Afonin S, Chernega AN, Rusanov EB, Platonov MO, Dubinina GG, Berditsch M, UIrich AS, Komarov IV. Conformationally rigid trifluoromethyl-substituted α-amino acid designed for peptide structure analysis by solid-state 19F NMR spectroscopy. Angew Chem Int Ed. 2006;45:5659–61.CrossRefGoogle Scholar
  17. 17.
    Tkachenko AN, Mykhailiuk PK, Afonin S, Radchenko DS, Kubyshkin VS, Ulrich AS, Komarov IV. A 19F NMR label to substitute polar amino acids in peptides: a CF3-substituted analogue of serine and threonine. Angew Chem Int Ed. 2013;52:1486–9.CrossRefGoogle Scholar
  18. 18.
    Kubyshkin VS, Mykhailiuk PK, Afonin S, Grage SL, Komarov IV, Ulrich AS. Incorporation of labile trans-4,5-difluoromethanoproline into a peptide as a stable label for 19F NMR structure analysis. J Fluor Chem. 2013;152:136–43.CrossRefGoogle Scholar
  19. 19.
    Tkachenko AN, Radchenko DS, Mykhailiuk PK, Afonin S, Ulrich AS, Komarov IV. Design, synthesis, and application of a trifluoromethylated phenylalanine analogue as a label to study peptides by solid-state 19F NMR spectroscopy. Angew Chem Int Ed. 2013;52:6504–7.CrossRefGoogle Scholar
  20. 20.
    Kubyshkin VS, Mykhailiuk PK, Afonin S, Ulrich AS, Komarov IV. Incorporation of cis- and trans-4,5-difluoromethanoprolines into polypeptides. Org Lett. 2012;14:5254–7.CrossRefGoogle Scholar
  21. 21.
    Kubyshkin VS, Komarov IV, Afonin S, Mykhailiuk PK, Grage SL, Ulrich AS. Trifluoromethyl-substituted α-amino acids as solid state 19F-NMR labels for structural studies of membrane-bound peptides. In: Gouverneur V, Müller K, editors. Fluorine in pharmaceutical and medicinal chemistry: from biophysical aspects to clinical applications. London: Imperial College Press; 2012. p. 91–138.CrossRefGoogle Scholar
  22. 22.
    Mykhailiuk PK, Afonin S, Palamarchuk GV, Shishkin OV, Ulrich AS, Komarov IV. Synthesis of trifluoromethyl-substituted proline analogues as 19F NMR labels for peptides in the polyproline II conformation. Angew Chem Int Ed. 2008;47:5765–7.CrossRefGoogle Scholar
  23. 23.
    Michurin OM, Afonin S, Berditsch M, Daniliuc CG, Ulrich AS, Komarov IV, Radchenko DS. Delivering structural information on the polar face of membrane-active peptides: 19F-NMR labels with a cationic side chain. Angew Chem Int Ed. 2016;55:14595–9.CrossRefGoogle Scholar
  24. 24.
    Maisch D, Wadhwani P, Afonin S, Böttcher C, Koksch B, Ulrich AS. Chemical labeling strategy with (R)- and (S)-triofluoromethylalanin for solid state 19F NMR analysis of peptaibols in membranes. J Am Chem Soc. 2009;131:15596–7.CrossRefGoogle Scholar
  25. 25.
    Afonin S, Dürr UHN, Glaser RW, Ulrich AS. ‘Boomerang’-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR. Magn Reson Chem. 2004;42:195–203.CrossRefGoogle Scholar
  26. 26.
    Wadhwani P, Strandberg E, van den Berg J, Mink C, Bürck J, Ciriello R, Ulrich AS. Dynamical structure of the short multifunctional peptide BP100 in membranes. Biochim Biophys Acta. 1838;2014:940–9.Google Scholar
  27. 27.
    Grasnick D, Sternberg U, Strandberg E, Wadhwani P, Ulrich AS. Irregular structure of the HIV fusion peptide in membranes demonstrated by solid-state NMR and MD simulations. Eur Biophys J. 2011;40:529–43.CrossRefGoogle Scholar
  28. 28.
    Grage SL, Kara S, Bordessa A, Doan V, Rizzolo F, Putzu M, Kubař T, Papini AM, Chaume G, Brigaud T, Afonin S, Ulrich AS. Orthogonal 19F-labeling for solid-state NMR reveals the conformation and orientation of short peptaibols in membranes. Submitted 2017.Google Scholar
  29. 29.
    Grage SL, Sani M-A, Cheneval O, Henriques ST, Schalck C, Heinzmann R, Mylne JS, Mykhailiuk PK, Afonin S, Komarov IV, Separovic F, Craik DJ, Ulrich AS. Orientation and location of the cyclotide kalata B1 in phospholipid bilayers revealed by solid-state NMR. Biophys J. 2017; in press.
  30. 30.
    Wadhwani P, Reichert J, Strandberg E, Bürck J, Misiewicz J, Afonin S, Heidenreich N, Fanghänel S, Mykhailiuk PK, Komarov IV, Ulrich AS. Stereochemical effects on the aggregation and biological properties of the fibril-forming peptide [KIGAKI]3 in membranes. Phys Chem Chem Phys. 2013;15:8962–71.CrossRefGoogle Scholar
  31. 31.
    Wadhwani P, Bürck J, Strandberg E, Mink C, Afonin S, Ulrich AS. Using a sterically restrictive amino acid as a 19F-NMR label to monitor and control peptide aggregation in membranes. J Am Chem Soc. 2008;130:16515–7.CrossRefGoogle Scholar
  32. 32.
    Strandberg E, Kanithasen N, Bürck J, Wadhwani P, Tiltak D, Zwernemann O, Ulrich AS. Solid state NMR analysis comparing the designer-made antibiotic MSI-103 with its parent peptide PGLa in lipid bilayers. Biochemistry. 2008;47:2601–16.CrossRefGoogle Scholar
  33. 33.
    Glaser RW, Sachse C, Dürr UHN, Afonin S, Wadhwani P, Strandberg E, Ulrich AS. Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR. Biophys J. 2005;88:3392–7.CrossRefGoogle Scholar
  34. 34.
    Afonin S, Grage SL, Ieronimo M, Wadhwani P, Ulrich AS. Temperature-dependent transmembrane insertion of the amphiphilic peptide PGLa in lipid bilayers observed by solid state 19F-NMR spectroscopy. J Am Chem Soc. 2008;130:16512–4.CrossRefGoogle Scholar
  35. 35.
    Afonin S, Mikhailiuk PK, Komarov IV, Ulrich AS. Evaluating the amino acid CF3-bicyclopentylglycine as a new label for solid-state 19F-NMR structure analysis of membrane-bound peptides. J Pept Sci. 2007;13:614–23.CrossRefGoogle Scholar
  36. 36.
    Radchenko DS, Kattge S, Kara S, Ulrich AS, Afonin S. Does a methionine-to-norleucine substitution in PGLa influence peptide-membrane interactions? Biochim Biophys Acta. 1858;2016:2019–27.Google Scholar
  37. 37.
    Mühlhäuser P, Wadhwani P, Strandberg E, Bürck J, Ulrich AS. Structural studies on the short DCD-derived antimicrobial peptide SSL-25. Submitted 2017.Google Scholar
  38. 38.
    Fanghänel S, Wadhwani P, Strandberg E, Verdurmen WPR, Bürck J, Ehni S, Mykhailiuk PK, Afonin S, Gerthsen D, Komarov IV, Brock R, Ulrich AS. Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state. PLoS One. 2014;9:e99653.CrossRefGoogle Scholar
  39. 39.
    Durell SR, Martin I, Ruysschaert JM, Shai Y, Blumenthal R. What studies of fusion peptides tell us about viral envelope glycoprotein-mediated membrane fusion (review). Mol Membr Biol. 1997;14:97–112.CrossRefGoogle Scholar
  40. 40.
    Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol. 2005;3:238–50.CrossRefGoogle Scholar
  41. 41.
    Soravia E, Martini G, Zasloff M. Antimicrobial properties of peptides from Xenopus granular gland secretions. FEBS Lett. 1988;228:337–40.CrossRefGoogle Scholar
  42. 42.
    Strandberg E, Wadhwani P, Tremouilhac P, Dürr UHN, Ulrich AS. Solid-state NMR analysis of the PGLa peptide orientation in DMPC bilayers: structural fidelity of 2H-labels versus high sensitivity of 19F-NMR. Biophys J. 2006;90:1676–86.CrossRefGoogle Scholar
  43. 43.
    Ieronimo M. Towards the activity of the antimicrobial peptide PGLa in cell membranes. Solid-state NMR studies of PGLa and magainin 2. PhD thesis: Institute of organic chemistry. Karlsruhe: University of Karlsruhe; 2008.Google Scholar
  44. 44.
    Maloy WL, Kari UP. Structure-activity studies on magainins and other host-defense peptides. Biopolymers. 1995;37:105–22.CrossRefGoogle Scholar
  45. 45.
    Toke O, O’Connor RD, Weldeghiorghis TK, Maloy WL, Glaser RW, Ulrich AS, Schaefer J. Structure of (KIAGKIA)3 aggregates in phospholipid bilayers by solid-state NMR. Biophys J. 2004;87:675–87.CrossRefGoogle Scholar
  46. 46.
    Bürck J, Roth S, Wadhwani P, Afonin S, Kanithasen N, Strandberg E, Ulrich AS. Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism. Biophys J. 2008;95:3872–81.CrossRefGoogle Scholar
  47. 47.
    Blazyk J, Wiegand R, Klein J, Hammer J, Epand RM, Epand RF, Maloy WL, Kari UP. A novel linear amphipathic β-sheet cationic antimicrobial peptide with enhanced selectivity for bacterial lipids. J Biol Chem. 2001;276:27899–906.CrossRefGoogle Scholar
  48. 48.
    Baechle D, Flad T, Cansier A, Steffen H, Schittek B, Tolson J, Herrmann T, Dihazi H, Beck A, Mueller GA, Mueller M, Stevanovic S, Garbe C, Mueller CA, Kalbacher H. Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L. J Biol Chem. 2006;281:5406–15.CrossRefGoogle Scholar
  49. 49.
    Leitgeb B, Szekeres A, Manczinger L, Vagvolgyi C, Kredics L. The history of alamethicin: a review of the most extensively studied peptaibol. Chem Biodivers. 2007;4:1027–51.CrossRefGoogle Scholar
  50. 50.
    Colgrave ML, Craik DJ. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry. 2004;43:5965–75.CrossRefGoogle Scholar
  51. 51.
    Langel Ü. Cell-penetrating peptides: processes and applications. Boca Raton: CRC Press; 2002.Google Scholar
  52. 52.
    Eggenberger K, Mink C, Wadhwani P, Ulrich AS, Nick P. Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells. ChemBioChem. 2011;12:132–7.CrossRefGoogle Scholar
  53. 53.
    Zamora-Carreras H, Strandberg E, Mühlhäuser P, Bürck J, Wadhwani P, Jiménez MÁ, Bruix M, Ulrich AS. Alanine scan and 2H NMR analysis of the membrane-active peptide BP100 point to a distinct carpet mechanism of action. Biochim Biophys Acta. 1858;2016:1328–38.Google Scholar
  54. 54.
    Oehlke J, Scheller A, Wiesner B, Krause E, Beyermann M, Klauschenz E, Melzig M, Bienert M. Cellular uptake of an α-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochim Biophys Acta. 1998;1414:127–39.CrossRefGoogle Scholar
  55. 55.
    Afonin S, Glaser RW, Berditchevskaia M, Wadhwani P, Guhrs KH, Mollmann U, Perner A, Ulrich AS. 4-Fluorophenylglycine as a label for 19F-NMR structure analysis of membrane-associated peptides. ChemBioChem. 2003;4:1151–63.CrossRefGoogle Scholar
  56. 56.
    Afonin S, Dürr UHN, Wadhwani P, Salgado JB, Ulrich AS. Solid state NMR structure analysis of the antimicrobial peptide gramicidin S in lipid membranes: concentration-dependent re-alignment and self-assembly as a β-barrel. Top Curr Chem. 2008;273:139–54.CrossRefGoogle Scholar
  57. 57.
    Afonin S, Glaser RW, Sachse C, Salgado J, Wadhwani P, Ulrich AS. 19F NMR screening of unrelated antimicrobial peptides shows that membrane interactions are largely governed by lipids. Biochim Biophys Acta. 1838;2014:2260–8.Google Scholar
  58. 58.
    Kubyshkin V, Afonin S, Kara S, Budisa N, Mykhailiuk PK, Ulrich AS. γ-(S)-Trifluoromethyl proline: evaluation as a structural substitute of proline for solid state 19F-NMR peptide studies. Org Biomol Chem. 2015;13:3171–81.CrossRefGoogle Scholar
  59. 59.
    Misiewicz J, Afonin S, Ulrich AS. Control and role of pH in peptide-lipid interactions in oriented membrane samples. Biochim Biophys Acta. 1848;2015:833–41.Google Scholar
  60. 60.
    Kokhan SO, Tymtsunik AV, Grage SL, Afonin S, Babii O, Berditsch M, Strizhak AV, Bandak D, Platonov MO, Komarov IV, Ulrich AS, Mykhailiuk PK. Design, synthesis, and application of an optimized monofluorinated aliphatic label for peptide studies by solid-state 19F NMR spectroscopy. Angew Chem Int Ed. 2016;55:14788–92.CrossRefGoogle Scholar
  61. 61.
    Zerweck J, Strandberg E, Bürck J, Reichert J, Wadhwani P, Kukharenko O, Ulrich AS. Homo- and heteromeric interaction strengths of the synergistic antimicrobial peptides PGLa and magainin 2 in membranes. Eur Biophys J. 2016;45:535–47.CrossRefGoogle Scholar
  62. 62.
    Strandberg E, Tremouilhac P, Wadhwani P, Ulrich AS. Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR. Biochim Biophys Acta. 1788;2009:1667–79.Google Scholar

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Biological Interfaces (IBG-2)Karlsruhe Institute of Technology (KIT)KarlsruheGermany
  2. 2.Institute of Organic ChemistryKITKarlsruheGermany

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