Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Nov;79(5):2644–2656. doi: 10.1016/S0006-3495(00)76503-9

The topology of lysine-containing amphipathic peptides in bilayers by circular dichroism, solid-state NMR, and molecular modeling.

B Vogt 1, P Ducarme 1, S Schinzel 1, R Brasseur 1, B Bechinger 1
PMCID: PMC1301145  PMID: 11053137

Abstract

In order to better understand the driving forces that determine the alignment of amphipathic helical polypeptides with respect to the surface of phospholipid bilayers, lysine-containing peptide sequences were designed, prepared by solid-phase chemical synthesis, and reconstituted into membranes. CD spectroscopy indicates that all peptides exhibit a high degree of helicity in the presence of SDS micelles or POPC small unilamellar vesicles. Proton-decoupled (31)P-NMR solid-state NMR spectroscopy demonstrates that in the presence of peptides liquid crystalline phosphatidylcholine membranes orient well along glass surfaces. The orientational distribution and dynamics of peptides labeled with (15)N at selected sites were investigated by proton-decoupled (15)N solid-state NMR spectroscopy. Polypeptides with a single lysine residue adopt a transmembrane orientation, thereby locating this polar amino acid within the core region of the bilayer. In contrast, peptides with > or = 3 lysines reside along the surface of the membrane. With 2 lysines in the center of an otherwise hydrophobic amino acid sequence the peptides assume a broad orientational distribution. The energy of lysine discharge, hydrophobic, polar, and all other interactions are estimated to quantitatively describe the polypeptide topologies observed. Furthermore, a molecular modeling algorithm based on the hydrophobicities of atoms in a continuous hydrophilic-hydrophobic-hydrophilic potential describes the experimentally observed peptide topologies well.

Full Text

The Full Text of this article is available as a PDF (457.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bechinger B., Kinder R., Helmle M., Vogt T. C., Harzer U., Schinzel S. Peptide structural analysis by solid-state NMR spectroscopy. Biopolymers. 1999;51(3):174–190. doi: 10.1002/(SICI)1097-0282(1999)51:3<174::AID-BIP2>3.0.CO;2-7. [DOI] [PubMed] [Google Scholar]
  2. Bechinger B., Ruysschaert J. M., Goormaghtigh E. Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra. Biophys J. 1999 Jan;76(1 Pt 1):552–563. doi: 10.1016/S0006-3495(99)77223-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bechinger B. Structure and functions of channel-forming peptides: magainins, cecropins, melittin and alamethicin. J Membr Biol. 1997 Apr 1;156(3):197–211. doi: 10.1007/s002329900201. [DOI] [PubMed] [Google Scholar]
  4. Bechinger B. The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy. Biochim Biophys Acta. 1999 Dec 15;1462(1-2):157–183. doi: 10.1016/s0005-2736(99)00205-9. [DOI] [PubMed] [Google Scholar]
  5. Bechinger B. Towards membrane protein design: pH-sensitive topology of histidine-containing polypeptides. J Mol Biol. 1996 Nov 15;263(5):768–775. doi: 10.1006/jmbi.1996.0614. [DOI] [PubMed] [Google Scholar]
  6. Bechinger B., Zasloff M., Opella S. J. Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy. Protein Sci. 1993 Dec;2(12):2077–2084. doi: 10.1002/pro.5560021208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Belrhali H., Nollert P., Royant A., Menzel C., Rosenbusch J. P., Landau E. M., Pebay-Peyroula E. Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution. Structure. 1999 Aug 15;7(8):909–917. doi: 10.1016/s0969-2126(99)80118-x. [DOI] [PubMed] [Google Scholar]
  8. Ben-Shaul A., Ben-Tal N., Honig B. Statistical thermodynamic analysis of peptide and protein insertion into lipid membranes. Biophys J. 1996 Jul;71(1):130–137. doi: 10.1016/S0006-3495(96)79208-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ben-Tal N., Ben-Shaul A., Nicholls A., Honig B. Free-energy determinants of alpha-helix insertion into lipid bilayers. Biophys J. 1996 Apr;70(4):1803–1812. doi: 10.1016/S0006-3495(96)79744-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bevins C. L., Zasloff M. Peptides from frog skin. Annu Rev Biochem. 1990;59:395–414. doi: 10.1146/annurev.bi.59.070190.002143. [DOI] [PubMed] [Google Scholar]
  11. Boman H. G. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol. 1995;13:61–92. doi: 10.1146/annurev.iy.13.040195.000425. [DOI] [PubMed] [Google Scholar]
  12. Brasseur R. Differentiation of lipid-associating helices by use of three-dimensional molecular hydrophobicity potential calculations. J Biol Chem. 1991 Aug 25;266(24):16120–16127. [PubMed] [Google Scholar]
  13. Cruciani R. A., Barker J. L., Durell S. R., Raghunathan G., Guy H. R., Zasloff M., Stanley E. F. Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes. Eur J Pharmacol. 1992 Aug 3;226(4):287–296. doi: 10.1016/0922-4106(92)90045-w. [DOI] [PubMed] [Google Scholar]
  14. Cruciani R. A., Barker J. L., Zasloff M., Chen H. C., Colamonici O. Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3792–3796. doi: 10.1073/pnas.88.9.3792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dempsey C. E. The actions of melittin on membranes. Biochim Biophys Acta. 1990 May 7;1031(2):143–161. doi: 10.1016/0304-4157(90)90006-x. [DOI] [PubMed] [Google Scholar]
  16. Doak D. G., Mulvey D., Kawaguchi K., Villalain J., Campbell I. D. Structural studies of synthetic peptides dissected from the voltage-gated sodium channel. J Mol Biol. 1996 May 17;258(4):672–687. doi: 10.1006/jmbi.1996.0278. [DOI] [PubMed] [Google Scholar]
  17. Ducarme P., Rahman M., Brasseur R. IMPALA: a simple restraint field to simulate the biological membrane in molecular structure studies. Proteins. 1998 Mar 1;30(4):357–371. [PubMed] [Google Scholar]
  18. Duclohier H., Molle G., Spach G. Antimicrobial peptide magainin I from Xenopus skin forms anion-permeable channels in planar lipid bilayers. Biophys J. 1989 Nov;56(5):1017–1021. doi: 10.1016/S0006-3495(89)82746-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Engelman D. M., Steitz T. A., Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986;15:321–353. doi: 10.1146/annurev.bb.15.060186.001541. [DOI] [PubMed] [Google Scholar]
  20. Gibson B. W., Poulter L., Williams D. H., Maggio J. E. Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis. J Biol Chem. 1986 Apr 25;261(12):5341–5349. [PubMed] [Google Scholar]
  21. Haimovich B., Tanaka J. C. Magainin-induced cytotoxicity in eukaryotic cells: kinetics, dose-response and channel characteristics. Biochim Biophys Acta. 1995 Dec 13;1240(2):149–158. doi: 10.1016/0005-2736(95)00204-9. [DOI] [PubMed] [Google Scholar]
  22. Hoffmann W., Richter K., Kreil G. A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin. EMBO J. 1983;2(5):711–714. doi: 10.1002/j.1460-2075.1983.tb01489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hultmark D. Drosophila as a model system for antibacterial peptides. Ciba Found Symp. 1994;186:107–122. doi: 10.1002/9780470514658.ch7. [DOI] [PubMed] [Google Scholar]
  24. Israelachvili J. N., Marcelja S., Horn R. G. Physical principles of membrane organization. Q Rev Biophys. 1980 May;13(2):121–200. doi: 10.1017/s0033583500001645. [DOI] [PubMed] [Google Scholar]
  25. Juretić D., Hendler R. W., Kamp F., Caughey W. S., Zasloff M., Westerhoff H. V. Magainin oligomers reversibly dissipate delta microH+ in cytochrome oxidase liposomes. Biochemistry. 1994 Apr 19;33(15):4562–4570. doi: 10.1021/bi00181a017. [DOI] [PubMed] [Google Scholar]
  26. Jähnig F. Thermodynamics and kinetics of protein incorporation into membranes. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3691–3695. doi: 10.1073/pnas.80.12.3691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ketchem R. R., Hu W., Cross T. A. High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. Science. 1993 Sep 10;261(5127):1457–1460. doi: 10.1126/science.7690158. [DOI] [PubMed] [Google Scholar]
  28. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  29. La Rocca P., Shai Y., Sansom M. S. Peptide-bilayer interactions: simulations of dermaseptin B, an antimicrobial peptide. Biophys Chem. 1999 Feb 1;76(2):145–159. doi: 10.1016/s0301-4622(98)00232-4. [DOI] [PubMed] [Google Scholar]
  30. Lambotte S., Jasperse P., Bechinger B. Orientational distribution of alpha-helices in the colicin B and E1 channel domains: a one and two dimensional 15N solid-state NMR investigation in uniaxially aligned phospholipid bilayers. Biochemistry. 1998 Jan 6;37(1):16–22. doi: 10.1021/bi9724671. [DOI] [PubMed] [Google Scholar]
  31. Lazo N. D., Hu W., Cross T. A. Low-temperature solid-state 15N NMR characterization of polypeptide backbone librations. J Magn Reson B. 1995 Apr;107(1):43–50. doi: 10.1006/jmrb.1995.1056. [DOI] [PubMed] [Google Scholar]
  32. Lewis B. A., Engelman D. M. Bacteriorhodopsin remains dispersed in fluid phospholipid bilayers over a wide range of bilayer thicknesses. J Mol Biol. 1983 May 15;166(2):203–210. doi: 10.1016/s0022-2836(83)80006-0. [DOI] [PubMed] [Google Scholar]
  33. Ludtke S. J., He K., Heller W. T., Harroun T. A., Yang L., Huang H. W. Membrane pores induced by magainin. Biochemistry. 1996 Oct 29;35(43):13723–13728. doi: 10.1021/bi9620621. [DOI] [PubMed] [Google Scholar]
  34. Ludtke S., He K., Huang H. Membrane thinning caused by magainin 2. Biochemistry. 1995 Dec 26;34(51):16764–16769. doi: 10.1021/bi00051a026. [DOI] [PubMed] [Google Scholar]
  35. Matsuzaki K., Murase O., Tokuda H., Funakoshi S., Fujii N., Miyajima K. Orientational and aggregational states of magainin 2 in phospholipid bilayers. Biochemistry. 1994 Mar 22;33(11):3342–3349. doi: 10.1021/bi00177a027. [DOI] [PubMed] [Google Scholar]
  36. Milik M., Skolnick J. Insertion of peptide chains into lipid membranes: an off-lattice Monte Carlo dynamics model. Proteins. 1993 Jan;15(1):10–25. doi: 10.1002/prot.340150104. [DOI] [PubMed] [Google Scholar]
  37. Monné M., Nilsson I., Johansson M., Elmhed N., von Heijne G. Positively and negatively charged residues have different effects on the position in the membrane of a model transmembrane helix. J Mol Biol. 1998 Dec 11;284(4):1177–1183. doi: 10.1006/jmbi.1998.2218. [DOI] [PubMed] [Google Scholar]
  38. Mouritsen O. G., Bloom M. Mattress model of lipid-protein interactions in membranes. Biophys J. 1984 Aug;46(2):141–153. doi: 10.1016/S0006-3495(84)84007-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. North C. L., Barranger-Mathys M., Cafiso D. S. Membrane orientation of the N-terminal segment of alamethicin determined by solid-state 15N NMR. Biophys J. 1995 Dec;69(6):2392–2397. doi: 10.1016/S0006-3495(95)80108-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ohsaki Y., Gazdar A. F., Chen H. C., Johnson B. E. Antitumor activity of magainin analogues against human lung cancer cell lines. Cancer Res. 1992 Jul 1;52(13):3534–3538. [PubMed] [Google Scholar]
  41. Picot D., Loll P. J., Garavito R. M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature. 1994 Jan 20;367(6460):243–249. doi: 10.1038/367243a0. [DOI] [PubMed] [Google Scholar]
  42. Sansom M. S. Alamethicin and related peptaibols--model ion channels. Eur Biophys J. 1993;22(2):105–124. doi: 10.1007/BF00196915. [DOI] [PubMed] [Google Scholar]
  43. Scherer P. G., Seelig J. Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles. Biochemistry. 1989 Sep 19;28(19):7720–7728. doi: 10.1021/bi00445a030. [DOI] [PubMed] [Google Scholar]
  44. Schümann M., Dathe M., Wieprecht T., Beyermann M., Bienert M. The tendency of magainin to associate upon binding to phospholipid bilayers. Biochemistry. 1997 Apr 8;36(14):4345–4351. doi: 10.1021/bi962304x. [DOI] [PubMed] [Google Scholar]
  45. Seelig J. 31P nuclear magnetic resonance and the head group structure of phospholipids in membranes. Biochim Biophys Acta. 1978 Jul 31;515(2):105–140. doi: 10.1016/0304-4157(78)90001-1. [DOI] [PubMed] [Google Scholar]
  46. Seelig J. Deuterium magnetic resonance: theory and application to lipid membranes. Q Rev Biophys. 1977 Aug;10(3):353–418. doi: 10.1017/s0033583500002948. [DOI] [PubMed] [Google Scholar]
  47. Segrest J. P., De Loof H., Dohlman J. G., Brouillette C. G., Anantharamaiah G. M. Amphipathic helix motif: classes and properties. Proteins. 1990;8(2):103–117. doi: 10.1002/prot.340080202. [DOI] [PubMed] [Google Scholar]
  48. Smith R., Separovic F., Milne T. J., Whittaker A., Bennett F. M., Cornell B. A., Makriyannis A. Structure and orientation of the pore-forming peptide, melittin, in lipid bilayers. J Mol Biol. 1994 Aug 19;241(3):456–466. doi: 10.1006/jmbi.1994.1520. [DOI] [PubMed] [Google Scholar]
  49. Soballe P. W., Maloy W. L., Myrga M. L., Jacob L. S., Herlyn M. Experimental local therapy of human melanoma with lytic magainin peptides. Int J Cancer. 1995 Jan 17;60(2):280–284. doi: 10.1002/ijc.2910600225. [DOI] [PubMed] [Google Scholar]
  50. Subczynski W. K., Lewis R. N., McElhaney R. N., Hodges R. S., Hyde J. S., Kusumi A. Molecular organization and dynamics of 1-palmitoyl-2-oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide. Biochemistry. 1998 Mar 3;37(9):3156–3164. doi: 10.1021/bi972148+. [DOI] [PubMed] [Google Scholar]
  51. Vaz Gomes A., de Waal A., Berden J. A., Westerhoff H. V. Electric potentiation, cooperativity, and synergism of magainin peptides in protein-free liposomes. Biochemistry. 1993 May 25;32(20):5365–5372. doi: 10.1021/bi00071a011. [DOI] [PubMed] [Google Scholar]
  52. Vogt T. C., Bechinger B. The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers. The effects of charges and pH. J Biol Chem. 1999 Oct 8;274(41):29115–29121. doi: 10.1074/jbc.274.41.29115. [DOI] [PubMed] [Google Scholar]
  53. White S. H., Wimley W. C. Membrane protein folding and stability: physical principles. Annu Rev Biophys Biomol Struct. 1999;28:319–365. doi: 10.1146/annurev.biophys.28.1.319. [DOI] [PubMed] [Google Scholar]
  54. Zhang Y. P., Lewis R. N., Henry G. D., Sykes B. D., Hodges R. S., McElhaney R. N. Peptide models of helical hydrophobic transmembrane segments of membrane proteins. 1. Studies of the conformation, intrabilayer orientation, and amide hydrogen exchangeability of Ac-K2-(LA)12-K2-amide. Biochemistry. 1995 Feb 21;34(7):2348–2361. doi: 10.1021/bi00007a031. [DOI] [PubMed] [Google Scholar]
  55. de Planque M. R., Kruijtzer J. A., Liskamp R. M., Marsh D., Greathouse D. V., Koeppe R. E., 2nd, de Kruijff B., Killian J. A. Different membrane anchoring positions of tryptophan and lysine in synthetic transmembrane alpha-helical peptides. J Biol Chem. 1999 Jul 23;274(30):20839–20846. doi: 10.1074/jbc.274.30.20839. [DOI] [PubMed] [Google Scholar]
  56. von Heijne G. Membrane proteins: the amino acid composition of membrane-penetrating segments. Eur J Biochem. 1981 Nov;120(2):275–278. doi: 10.1111/j.1432-1033.1981.tb05700.x. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES