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. 1995 Nov;69(5):1999–2010. doi: 10.1016/S0006-3495(95)80070-6

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length.

T D Bradrick 1, A Philippetis 1, S Georghiou 1
PMCID: PMC1236433  PMID: 8580343

Abstract

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.

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1999

Selected References

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  1. Altenbach C., Froncisz W., Hyde J. S., Hubbell W. L. Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance. Biophys J. 1989 Dec;56(6):1183–1191. doi: 10.1016/S0006-3495(89)82765-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altenbach C., Hubbell W. L. The aggregation state of spin-labeled melittin in solution and bound to phospholipid membranes: evidence that membrane-bound melittin is monomeric. Proteins. 1988;3(4):230–242. doi: 10.1002/prot.340030404. [DOI] [PubMed] [Google Scholar]
  3. Beschiaschvili G., Seelig J. Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes. Biochemistry. 1990 Jan 9;29(1):52–58. doi: 10.1021/bi00453a007. [DOI] [PubMed] [Google Scholar]
  4. Bloom M., Evans E., Mouritsen O. G. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q Rev Biophys. 1991 Aug;24(3):293–397. doi: 10.1017/s0033583500003735. [DOI] [PubMed] [Google Scholar]
  5. Bradrick T. D., Dasseux J. L., Abdalla M., Aminzadeh A., Georghiou S. Effects of bee venom melittin on the order and dynamics of dimyristoylphosphatidylcholine unilamellar and multilamellar vesicles. Biochim Biophys Acta. 1987 Jun 12;900(1):17–26. doi: 10.1016/0005-2736(87)90273-2. [DOI] [PubMed] [Google Scholar]
  6. Bradrick T. D., Freire E., Georghiou S. A high-sensitivity differential scanning calorimetric study of the interaction of melittin with dipalmitoylphosphatidylcholine fused unilamellar vesicles. Biochim Biophys Acta. 1989 Jun 26;982(1):94–102. doi: 10.1016/0005-2736(89)90179-x. [DOI] [PubMed] [Google Scholar]
  7. Bradrick T. D., Georghiou S. Kinetics of melittin-induced fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles. Biochim Biophys Acta. 1987 Dec 11;905(2):494–498. doi: 10.1016/0005-2736(87)90479-2. [DOI] [PubMed] [Google Scholar]
  8. Dasseux J. L., Faucon J. F., Lafleur M., Pezolet M., Dufourcq J. A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids. Biochim Biophys Acta. 1984 Aug 8;775(1):37–50. doi: 10.1016/0005-2736(84)90232-3. [DOI] [PubMed] [Google Scholar]
  9. Dawson C. R., Drake A. F., Helliwell J., Hider R. C. The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta. 1978 Jun 16;510(1):75–86. doi: 10.1016/0005-2736(78)90131-1. [DOI] [PubMed] [Google Scholar]
  10. DeGrado W. F., Musso G. F., Lieber M., Kaiser E. T., Kézdy F. J. Kinetics and mechanism of hemolysis induced by melittin and by a synthetic melittin analogue. Biophys J. 1982 Jan;37(1):329–338. doi: 10.1016/S0006-3495(82)84681-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dempsey C. E., Butler G. S. Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ. Biochemistry. 1992 Dec 8;31(48):11973–11977. doi: 10.1021/bi00163a003. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Dempsey C. E., Watts A. A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2. Biochemistry. 1987 Sep 8;26(18):5803–5811. doi: 10.1021/bi00392a033. [DOI] [PubMed] [Google Scholar]
  14. Drake A. F., Hider R. C. The structure of melittin in lipid bilayer membranes. Biochim Biophys Acta. 1979 Aug 7;555(2):371–373. doi: 10.1016/0005-2736(79)90178-0. [DOI] [PubMed] [Google Scholar]
  15. Dufourc E. J., Smith I. C., Dufourcq J. Molecular details of melittin-induced lysis of phospholipid membranes as revealed by deuterium and phosphorus NMR. Biochemistry. 1986 Oct 21;25(21):6448–6455. doi: 10.1021/bi00369a016. [DOI] [PubMed] [Google Scholar]
  16. Eytan G. D., Almary T. Melittin-induced fusion of acidic liposomes. FEBS Lett. 1983 May 30;156(1):29–32. doi: 10.1016/0014-5793(83)80241-5. [DOI] [PubMed] [Google Scholar]
  17. Faucon J. F., Dufourcq J., Lussan C. The self-association of melittin and its binding to lipids: an intrinsic fluorescence polarization study. FEBS Lett. 1979 Jun 1;102(1):187–190. doi: 10.1016/0014-5793(79)80956-4. [DOI] [PubMed] [Google Scholar]
  18. Freire E., Biltonen R. Estimation of molecular averages and equilibrium fluctuations in lipid bilayer systems from the excess heat capacity function. Biochim Biophys Acta. 1978 Dec 4;514(1):54–68. doi: 10.1016/0005-2736(78)90076-7. [DOI] [PubMed] [Google Scholar]
  19. Frey S., Tamm L. K. Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study. Biophys J. 1991 Oct;60(4):922–930. doi: 10.1016/S0006-3495(91)82126-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garnier J., Gaye P., Mercier J. C., Robson B. Structural properties of signal peptides and their membrane insertion. Biochimie. 1980;62(4):231–239. doi: 10.1016/s0300-9084(80)80397-x. [DOI] [PubMed] [Google Scholar]
  21. Georghiou S., Thompson M., Mukhopadhyay A. K. Melittin-phospholipid interaction studied by employing the single tryptophan residue as an intrinsic fluorescent probe. Biochim Biophys Acta. 1982 Jun 14;688(2):441–452. doi: 10.1016/0005-2736(82)90355-8. [DOI] [PubMed] [Google Scholar]
  22. Hermetter A., Lakowicz J. R. The aggregation state of mellitin in lipid bilayers. An energy transfer study. J Biol Chem. 1986 Jun 25;261(18):8243–8248. [PubMed] [Google Scholar]
  23. Huang C. Studies on phosphatidylcholine vesicles. Formation and physical characteristics. Biochemistry. 1969 Jan;8(1):344–352. doi: 10.1021/bi00829a048. [DOI] [PubMed] [Google Scholar]
  24. Hubbell W. L., McConnell H. M. Molecular motion in spin-labeled phospholipids and membranes. J Am Chem Soc. 1971 Jan 27;93(2):314–326. doi: 10.1021/ja00731a005. [DOI] [PubMed] [Google Scholar]
  25. Ipsen J. H., Jørgensen K., Mouritsen O. G. Density fluctuations in saturated phospholipid bilayers increase as the acyl-chain length decreases. Biophys J. 1990 Nov;58(5):1099–1107. doi: 10.1016/S0006-3495(90)82452-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Israelachvili J. N. Refinement of the fluid-mosaic model of membrane structure. Biochim Biophys Acta. 1977 Sep 5;469(2):221–225. doi: 10.1016/0005-2736(77)90185-7. [DOI] [PubMed] [Google Scholar]
  27. John E., Jähnig F. Aggregation state of melittin in lipid vesicle membranes. Biophys J. 1991 Aug;60(2):319–328. doi: 10.1016/S0006-3495(91)82056-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jähnig F. Critical effects from lipid-protein interaction in membranes. I. Theoretical description. Biophys J. 1981 Nov;36(2):329–345. doi: 10.1016/S0006-3495(81)84735-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kaiser E. T., Kézdy F. J. Secondary structures of proteins and peptides in amphiphilic environments. (A review). Proc Natl Acad Sci U S A. 1983 Feb;80(4):1137–1143. doi: 10.1073/pnas.80.4.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Knöppel E., Eisenberg D., Wickner W. Interactions of melittin, a preprotein model, with detergents. Biochemistry. 1979 Sep 18;18(19):4177–4181. doi: 10.1021/bi00586a021. [DOI] [PubMed] [Google Scholar]
  31. Kuchinka E., Seelig J. Interaction of melittin with phosphatidylcholine membranes. Binding isotherm and lipid head-group conformation. Biochemistry. 1989 May 16;28(10):4216–4221. doi: 10.1021/bi00436a014. [DOI] [PubMed] [Google Scholar]
  32. Lis L. J., McAlister M., Fuller N., Rand R. P., Parsegian V. A. Measurement of the lateral compressibility of several phospholipid bilayers. Biophys J. 1982 Mar;37(3):667–672. [PMC free article] [PubMed] [Google Scholar]
  33. Marcelja S. Lipid-mediated protein interaction in membranes. Biochim Biophys Acta. 1976 Nov 11;455(1):1–7. doi: 10.1016/0005-2736(76)90149-8. [DOI] [PubMed] [Google Scholar]
  34. Marsh D., Watts A., Knowles P. F. Evidence for phase boundary lipid. Permeability of Tempo-choline into dimyristoylphosphatidylcholine vesicles at the phase transition. Biochemistry. 1976 Aug 10;15(16):3570–3578. doi: 10.1021/bi00661a027. [DOI] [PubMed] [Google Scholar]
  35. Mollay C., Kreil G. Enhancement of bee venom phospholipase A2 activity by melittin, direct lytic factor from cobra venom and polymyxin B. FEBS Lett. 1974 Sep 15;46(1):141–144. doi: 10.1016/0014-5793(74)80354-6. [DOI] [PubMed] [Google Scholar]
  36. Morgan C. G., Williamson H., Fuller S., Hudson B. Melittin induces fusion of unilamellar phospholipid vesicles. Biochim Biophys Acta. 1983 Aug 10;732(3):668–674. doi: 10.1016/0005-2736(83)90245-6. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Myers M., Mayorga O. L., Emtage J., Freire E. Thermodynamic characterization of interactions between ornithine transcarbamylase leader peptide and phospholipid bilayer membranes. Biochemistry. 1987 Jul 14;26(14):4309–4315. doi: 10.1021/bi00388a019. [DOI] [PubMed] [Google Scholar]
  39. Ohki S., Marcus E., Sukumaran D. K., Arnold K. Interaction of melittin with lipid membranes. Biochim Biophys Acta. 1994 Sep 14;1194(2):223–232. doi: 10.1016/0005-2736(94)90303-4. [DOI] [PubMed] [Google Scholar]
  40. Otto M. R., Lillo M. P., Beechem J. M. Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments. Biophys J. 1994 Dec;67(6):2511–2521. doi: 10.1016/S0006-3495(94)80741-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Owicki J. C., Springgate M. W., McConnell H. M. Theoretical study of protein--lipid interactions in bilayer membranes. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1616–1619. doi: 10.1073/pnas.75.4.1616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Papahadjopoulos D., Jacobson K., Nir S., Isac T. Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol. Biochim Biophys Acta. 1973 Jul 6;311(3):330–348. doi: 10.1016/0005-2736(73)90314-3. [DOI] [PubMed] [Google Scholar]
  43. Prendergast F. G., Haugland R. P., Callahan P. J. 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers. Biochemistry. 1981 Dec 22;20(26):7333–7338. doi: 10.1021/bi00529a002. [DOI] [PubMed] [Google Scholar]
  44. Quay S. C., Condie C. C. Conformational studies of aqueous melittin: thermodynamic parameters of the monomer-tetramer self-association reaction. Biochemistry. 1983 Feb 1;22(3):695–700. doi: 10.1021/bi00272a026. [DOI] [PubMed] [Google Scholar]
  45. Sansom M. S. The biophysics of peptide models of ion channels. Prog Biophys Mol Biol. 1991;55(3):139–235. doi: 10.1016/0079-6107(91)90004-c. [DOI] [PubMed] [Google Scholar]
  46. Schwarz G., Beschiaschvili G. Thermodynamic and kinetic studies on the association of melittin with a phospholipid bilayer. Biochim Biophys Acta. 1989 Feb 13;979(1):82–90. doi: 10.1016/0005-2736(89)90526-9. [DOI] [PubMed] [Google Scholar]
  47. Schwarz G., Zong R. T., Popescu T. Kinetics of melittin induced pore formation in the membrane of lipid vesicles. Biochim Biophys Acta. 1992 Sep 21;1110(1):97–104. doi: 10.1016/0005-2736(92)90299-2. [DOI] [PubMed] [Google Scholar]
  48. Seelig J., Seelig A. Lipid conformation in model membranes and biological membranes. Q Rev Biophys. 1980 Feb;13(1):19–61. doi: 10.1017/s0033583500000305. [DOI] [PubMed] [Google Scholar]
  49. Sekharam K. M., Bradrick T. D., Georghiou S. Kinetics of melittin binding to phospholipid small unilamellar vesicles. Biochim Biophys Acta. 1991 Mar 18;1063(1):171–174. doi: 10.1016/0005-2736(91)90367-h. [DOI] [PubMed] [Google Scholar]
  50. Sessa G., Freer J. H., Colacicco G., Weissmann G. Interaction of alytic polypeptide, melittin, with lipid membrane systems. J Biol Chem. 1969 Jul 10;244(13):3575–3582. [PubMed] [Google Scholar]
  51. Sperotto M. M., Mouritsen O. G. Monte Carlo simulation studies of lipid order parameter profiles near integral membrane proteins. Biophys J. 1991 Feb;59(2):261–270. doi: 10.1016/S0006-3495(91)82219-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Strom R., Crifo C., Viti V., Guidoni L., Podo F. Variations in circular dichroism and proton-NMR relaxation properties of melittin upon interaction with phospholipids. FEBS Lett. 1978 Dec 1;96(1):45–50. doi: 10.1016/0014-5793(78)81059-x. [DOI] [PubMed] [Google Scholar]
  53. Stubbs C. D., Kinosita K., Jr, Munkonge F., Quinn P. J., Ikegami A. The dynamics of lipid motion in sarcoplasmic reticulum membranes determined by steady-state and time-resolved fluorescence measurements on 1,6-diphenyl-1,3,5-hexatriene and related molecules. Biochim Biophys Acta. 1984 Sep 5;775(3):374–380. doi: 10.1016/0005-2736(84)90193-7. [DOI] [PubMed] [Google Scholar]
  54. Szoka F., Jr, Papahadjopoulos D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu Rev Biophys Bioeng. 1980;9:467–508. doi: 10.1146/annurev.bb.09.060180.002343. [DOI] [PubMed] [Google Scholar]
  55. Talbot J. C., Faucon J. F., Dufourcq J. Different states of self-association of melittin in phospholipid bilayers. A resonance energy transfer approach. Eur Biophys J. 1987;15(3):147–157. doi: 10.1007/BF00263679. [DOI] [PubMed] [Google Scholar]
  56. Terwilliger T. C., Weissman L., Eisenberg D. The structure of melittin in the form I crystals and its implication for melittin's lytic and surface activities. Biophys J. 1982 Jan;37(1):353–361. doi: 10.1016/S0006-3495(82)84683-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tosteson M. T., Alvarez O., Hubbell W., Bieganski R. M., Attenbach C., Caporales L. H., Levy J. J., Nutt R. F., Rosenblatt M., Tosteson D. C. Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels. Biophys J. 1990 Dec;58(6):1367–1375. doi: 10.1016/S0006-3495(90)82483-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vogel H. Incorporation of melittin into phosphatidylcholine bilayers. Study of binding and conformational changes. FEBS Lett. 1981 Nov 2;134(1):37–42. doi: 10.1016/0014-5793(81)80545-5. [DOI] [PubMed] [Google Scholar]
  59. Vogel H., Jähnig F., Hoffmann V., Stümpel J. The orientation of melittin in lipid membranes. A polarized infrared spectroscopy study. Biochim Biophys Acta. 1983 Sep 7;733(2):201–209. doi: 10.1016/0005-2736(83)90523-0. [DOI] [PubMed] [Google Scholar]
  60. Vogel H., Jähnig F. The structure of melittin in membranes. Biophys J. 1986 Oct;50(4):573–582. doi: 10.1016/S0006-3495(86)83497-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wiener M. C., White S. H. Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. III. Complete structure. Biophys J. 1992 Feb;61(2):434–447. doi: 10.1016/S0006-3495(92)81849-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yunes R., Goldhammer A. R., Garner W. K., Cordes E. H. Phospholipases: melittin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes. Arch Biochem Biophys. 1977 Sep;183(1):105–112. doi: 10.1016/0003-9861(77)90424-6. [DOI] [PubMed] [Google Scholar]
  63. van der Heide U. A., Levine Y. K. A computer simulation study of the relation between lipid and probe behaviour in bilayer systems. Biochim Biophys Acta. 1994 Oct 12;1195(1):1–10. doi: 10.1016/0005-2736(94)90002-7. [DOI] [PubMed] [Google Scholar]

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