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. 2001 Jul;81(1):285–304. doi: 10.1016/S0006-3495(01)75699-8

Interaction of synthetic HA2 influenza fusion peptide analog with model membranes.

D V Zhelev 1, N Stoicheva 1, P Scherrer 1, D Needham 1
PMCID: PMC1301511  PMID: 11423414

Abstract

The interaction of the synthetic 21 amino acid peptide (AcE4K) with 1-oleoyl-2-[caproyl-7-NBD]-sn-glycero-3-phosphocholine membranes is used as a model system for the pH-sensitive binding of fusion peptides to membranes. The sequence of AcE4K (Ac-GLFEAIAGFIENGWEGMIDGK) is based on the sequence of the hemagglutinin HA2 fusion peptide and has similar partitioning into phosphatidylcholine membranes as the viral peptide. pH-dependent partitioning in the membrane, circular dichroism, tryptophan fluorescence, change of membrane area, and membrane strength, are measured to characterize various key aspects of the peptide-membrane interaction. The experimental results show that the partitioning of AcE4K in the membrane is pH dependent. The bound peptide inserts in the membrane, which increases the overall membrane area in a pH-dependent manner, however the depth of insertion of the peptide in the membrane is independent of pH. This result suggests that the binding of the peptide to the membrane is driven by the protonation of its three glutamatic acids and the aspartic acid, which results in an increase of the number of bound molecules as the pH decreases from pH 7 to 4.5. The transition between the bound state and the free state is characterized by the Gibbs energy for peptide binding. This Gibbs energy for pH 5 is equal to -30.2 kJ/mol (-7.2 kcal/mol). Most of the change of the Gibbs energy during the binding of AcE4K is due to the enthalpy of binding -27.3 kJ/mol (-6.5 kcal/mol), while the entropy change is relatively small and is on the order of 6.4 J/mol.K (2.3 cal/mol.K). The energy barrier separating the bound and the free state, is characterized by the Gibbs energy of the transition state for peptide adsorption. This Gibbs energy is equal to 51.3 kJ/mol (12.3 kcal/mol). The insertion of the peptide into the membrane is coupled with work for creation of a vacancy for the peptide in the membrane. This work is calculated from the measured area occupied by a single peptide molecule (220 A(2)) and the membrane elasticity (190 mN/m), and is equal to 15.5 kJ/mol (3.7 kcal/mol). The comparison of the work for creating a vacancy and the Gibbs energy of the transition state shows that the work for creating a vacancy may have significant effect on the rate of peptide insertion and therefore plays an important role in peptide binding. Because the work for creating a vacancy depends on membrane elasticity and the elasticity of the membrane is dependent on membrane composition, this provides a tool for modulating the pH for membrane instability by changing membrane composition. The insertion of the peptide in the membrane does not affect the membrane permeability for water, which shows that the peptide does not perturb substantially the packing of the hydrocarbon region. However, the ability of the membrane to retain solutes in the presence of peptide is compromised, suggesting that the inserted peptide promotes formation of short living pores. The integrity of the membrane is substantially compromised below pH 4.8 (threshold pH), when large pores are formed and the membrane breaks down. The binding of the peptide in the pore region is reversible, and the pore size varies on the experimental conditions, which suggests that the peptide in the pore region does not form oligomers.

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Selected References

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  1. Alford D., Ellens H., Bentz J. Fusion of influenza virus with sialic acid-bearing target membranes. Biochemistry. 1994 Mar 1;33(8):1977–1987. doi: 10.1021/bi00174a002. [DOI] [PubMed] [Google Scholar]
  2. Bailey A. L., Monck M. A., Cullis P. R. pH-induced destabilization of lipid bilayers by a lipopeptide derived from influenza hemagglutinin. Biochim Biophys Acta. 1997 Mar 13;1324(2):232–244. doi: 10.1016/s0005-2736(96)00228-3. [DOI] [PubMed] [Google Scholar]
  3. Bentz J., Ellens H., Alford D. An architecture for the fusion site of influenza hemagglutinin. FEBS Lett. 1990 Dec 10;276(1-2):1–5. doi: 10.1016/0014-5793(90)80492-2. [DOI] [PubMed] [Google Scholar]
  4. Bentz J. Membrane fusion mediated by coiled coils: a hypothesis. Biophys J. 2000 Feb;78(2):886–900. doi: 10.1016/S0006-3495(00)76646-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bullough P. A., Hughson F. M., Skehel J. J., Wiley D. C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994 Sep 1;371(6492):37–43. doi: 10.1038/371037a0. [DOI] [PubMed] [Google Scholar]
  6. Burger K. N., Wharton S. A., Demel R. A., Verkleij A. J. The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs. Biochim Biophys Acta. 1991 Jun 18;1065(2):121–129. doi: 10.1016/0005-2736(91)90221-s. [DOI] [PubMed] [Google Scholar]
  7. Chang C. T., Wu C. S., Yang J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem. 1978 Nov;91(1):13–31. doi: 10.1016/0003-2697(78)90812-6. [DOI] [PubMed] [Google Scholar]
  8. Chattopadhyay A., London E. Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry. 1987 Jan 13;26(1):39–45. doi: 10.1021/bi00375a006. [DOI] [PubMed] [Google Scholar]
  9. Chernomordik L. V., Frolov V. A., Leikina E., Bronk P., Zimmerberg J. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J Cell Biol. 1998 Mar 23;140(6):1369–1382. doi: 10.1083/jcb.140.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Danieli T., Pelletier S. L., Henis Y. I., White J. M. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J Cell Biol. 1996 May;133(3):559–569. doi: 10.1083/jcb.133.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Durrer P., Galli C., Hoenke S., Corti C., Glück R., Vorherr T., Brunner J. H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region. J Biol Chem. 1996 Jun 7;271(23):13417–13421. doi: 10.1074/jbc.271.23.13417. [DOI] [PubMed] [Google Scholar]
  12. Fattal E., Nir S., Parente R. A., Szoka F. C., Jr Pore-forming peptides induce rapid phospholipid flip-flop in membranes. Biochemistry. 1994 May 31;33(21):6721–6731. doi: 10.1021/bi00187a044. [DOI] [PubMed] [Google Scholar]
  13. Gaudin Y., Ruigrok R. W., Brunner J. Low-pH induced conformational changes in viral fusion proteins: implications for the fusion mechanism. J Gen Virol. 1995 Jul;76(Pt 7):1541–1556. doi: 10.1099/0022-1317-76-7-1541. [DOI] [PubMed] [Google Scholar]
  14. Gething M. J., Doms R. W., York D., White J. Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J Cell Biol. 1986 Jan;102(1):11–23. doi: 10.1083/jcb.102.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gray C., Tatulian S. A., Wharton S. A., Tamm L. K. Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers. Biophys J. 1996 May;70(5):2275–2286. doi: 10.1016/S0006-3495(96)79793-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harter C., James P., Bächi T., Semenza G., Brunner J. Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide". J Biol Chem. 1989 Apr 15;264(11):6459–6464. [PubMed] [Google Scholar]
  17. Ishiguro R., Matsumoto M., Takahashi S. Interaction of fusogenic synthetic peptide with phospholipid bilayers: orientation of the peptide alpha-helix and binding isotherm. Biochemistry. 1996 Apr 16;35(15):4976–4983. doi: 10.1021/bi952547+. [DOI] [PubMed] [Google Scholar]
  18. Kemble G. W., Danieli T., White J. M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell. 1994 Jan 28;76(2):383–391. doi: 10.1016/0092-8674(94)90344-1. [DOI] [PubMed] [Google Scholar]
  19. Kwok R., Evans E. Thermoelasticity of large lecithin bilayer vesicles. Biophys J. 1981 Sep;35(3):637–652. doi: 10.1016/S0006-3495(81)84817-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lakowicz J. R., Keating S. Binding of an indole derivative to micelles as quantified by phase-sensitive detection of fluorescence. J Biol Chem. 1983 May 10;258(9):5519–5524. [PubMed] [Google Scholar]
  21. Lear J. D., DeGrado W. F. Membrane binding and conformational properties of peptides representing the NH2 terminus of influenza HA-2. J Biol Chem. 1987 May 15;262(14):6500–6505. [PubMed] [Google Scholar]
  22. Lewis J. R., Cafiso D. S. Correlation between the free energy of a channel-forming voltage-gated peptide and the spontaneous curvature of bilayer lipids. Biochemistry. 1999 May 4;38(18):5932–5938. doi: 10.1021/bi9828167. [DOI] [PubMed] [Google Scholar]
  23. Longo M. L., Waring A. J., Hammer D. A. Interaction of the influenza hemagglutinin fusion peptide with lipid bilayers: area expansion and permeation. Biophys J. 1997 Sep;73(3):1430–1439. doi: 10.1016/S0006-3495(97)78175-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lundbaek J. A., Andersen O. S. Spring constants for channel-induced lipid bilayer deformations. Estimates using gramicidin channels. Biophys J. 1999 Feb;76(2):889–895. doi: 10.1016/S0006-3495(99)77252-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lundbaek J. A., Maer A. M., Andersen O. S. Lipid bilayer electrostatic energy, curvature stress, and assembly of gramicidin channels. Biochemistry. 1997 May 13;36(19):5695–5701. doi: 10.1021/bi9619841. [DOI] [PubMed] [Google Scholar]
  27. Lüneberg J., Martin I., Nüssler F., Ruysschaert J. M., Herrmann A. Structure and topology of the influenza virus fusion peptide in lipid bilayers. J Biol Chem. 1995 Nov 17;270(46):27606–27614. doi: 10.1074/jbc.270.46.27606. [DOI] [PubMed] [Google Scholar]
  28. Macosko J. C., Kim C. H., Shin Y. K. The membrane topology of the fusion peptide region of influenza hemagglutinin determined by spin-labeling EPR. J Mol Biol. 1997 Apr 18;267(5):1139–1148. doi: 10.1006/jmbi.1997.0931. [DOI] [PubMed] [Google Scholar]
  29. Matlin K. S., Reggio H., Helenius A., Simons K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 1981 Dec;91(3 Pt 1):601–613. doi: 10.1083/jcb.91.3.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McIntosh T. J., Simon S. A. Hydration force and bilayer deformation: a reevaluation. Biochemistry. 1986 Jul 15;25(14):4058–4066. doi: 10.1021/bi00362a011. [DOI] [PubMed] [Google Scholar]
  31. Melikyan G. B., White J. M., Cohen F. S. GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes. J Cell Biol. 1995 Nov;131(3):679–691. doi: 10.1083/jcb.131.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Needham D., Nunn R. S. Elastic deformation and failure of lipid bilayer membranes containing cholesterol. Biophys J. 1990 Oct;58(4):997–1009. doi: 10.1016/S0006-3495(90)82444-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Needham D., Stoicheva N., Zhelev D. V. Exchange of monooleoylphosphatidylcholine as monomer and micelle with membranes containing poly(ethylene glycol)-lipid. Biophys J. 1997 Nov;73(5):2615–2629. doi: 10.1016/S0006-3495(97)78291-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Needham D., Zhelev D. V. Lysolipid exchange with lipid vesicle membranes. Ann Biomed Eng. 1995 May-Jun;23(3):287–298. doi: 10.1007/BF02584429. [DOI] [PubMed] [Google Scholar]
  35. Nielsen C., Goulian M., Andersen O. S. Energetics of inclusion-induced bilayer deformations. Biophys J. 1998 Apr;74(4):1966–1983. doi: 10.1016/S0006-3495(98)77904-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Olbrich K., Rawicz W., Needham D., Evans E. Water permeability and mechanical strength of polyunsaturated lipid bilayers. Biophys J. 2000 Jul;79(1):321–327. doi: 10.1016/S0006-3495(00)76294-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rafalski M., Ortiz A., Rockwell A., van Ginkel L. C., Lear J. D., DeGrado W. F., Wilschut J. Membrane fusion activity of the influenza virus hemagglutinin: interaction of HA2 N-terminal peptides with phospholipid vesicles. Biochemistry. 1991 Oct 22;30(42):10211–10220. doi: 10.1021/bi00106a020. [DOI] [PubMed] [Google Scholar]
  38. Rogers G. N., Paulson J. C., Daniels R. S., Skehel J. J., Wilson I. A., Wiley D. C. Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature. 1983 Jul 7;304(5921):76–78. doi: 10.1038/304076a0. [DOI] [PubMed] [Google Scholar]
  39. Rohl C. A., Chakrabartty A., Baldwin R. L. Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol. Protein Sci. 1996 Dec;5(12):2623–2637. doi: 10.1002/pro.5560051225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ruigrok R. W., Aitken A., Calder L. J., Martin S. R., Skehel J. J., Wharton S. A., Weis W., Wiley D. C. Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion. J Gen Virol. 1988 Nov;69(Pt 11):2785–2795. doi: 10.1099/0022-1317-69-11-2785. [DOI] [PubMed] [Google Scholar]
  41. Sauter N. K., Bednarski M. D., Wurzburg B. A., Hanson J. E., Whitesides G. M., Skehel J. J., Wiley D. C. Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study. Biochemistry. 1989 Oct 17;28(21):8388–8396. doi: 10.1021/bi00447a018. [DOI] [PubMed] [Google Scholar]
  42. Schroth-Diez B., Ponimaskin E., Reverey H., Schmidt M. F., Herrmann A. Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin. J Virol. 1998 Jan;72(1):133–141. doi: 10.1128/jvi.72.1.133-141.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Seelig J. Titration calorimetry of lipid-peptide interactions. Biochim Biophys Acta. 1997 Mar 14;1331(1):103–116. doi: 10.1016/s0304-4157(97)00002-6. [DOI] [PubMed] [Google Scholar]
  44. Shangguan T., Alford D., Bentz J. Influenza-virus-liposome lipid mixing is leaky and largely insensitive to the material properties of the target membrane. Biochemistry. 1996 Apr 16;35(15):4956–4965. doi: 10.1021/bi9526903. [DOI] [PubMed] [Google Scholar]
  45. Stegmann T., Hoekstra D., Scherphof G., Wilschut J. Kinetics of pH-dependent fusion between influenza virus and liposomes. Biochemistry. 1985 Jun 18;24(13):3107–3113. doi: 10.1021/bi00334a006. [DOI] [PubMed] [Google Scholar]
  46. Steinhauer D. A., Wharton S. A., Skehel J. J., Wiley D. C. Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin. J Virol. 1995 Nov;69(11):6643–6651. doi: 10.1128/jvi.69.11.6643-6651.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Takahashi S. Conformation of membrane fusion-active 20-residue peptides with or without lipid bilayers. Implication of alpha-helix formation for membrane fusion. Biochemistry. 1990 Jul 3;29(26):6257–6264. doi: 10.1021/bi00478a021. [DOI] [PubMed] [Google Scholar]
  48. Weber T., Paesold G., Galli C., Mischler R., Semenza G., Brunner J. Evidence for H(+)-induced insertion of influenza hemagglutinin HA2 N-terminal segment into viral membrane. J Biol Chem. 1994 Jul 15;269(28):18353–18358. [PubMed] [Google Scholar]
  49. Wimley W. C., White S. H. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat Struct Biol. 1996 Oct;3(10):842–848. doi: 10.1038/nsb1096-842. [DOI] [PubMed] [Google Scholar]
  50. Zhelev D. V. Exchange of monooleoylphosphatidylcholine with single egg phosphatidylcholine vesicle membranes. Biophys J. 1996 Jul;71(1):257–273. doi: 10.1016/S0006-3495(96)79222-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou Z., Macosko J. C., Hughes D. W., Sayer B. G., Hawes J., Epand R. M. 15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions. Biophys J. 2000 May;78(5):2418–2425. doi: 10.1016/S0006-3495(00)76785-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

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