Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Aug;75(2):840–852. doi: 10.1016/S0006-3495(98)77573-3

The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

P Lasch 1, C P Schultz 1, D Naumann 1
PMCID: PMC1299758  PMID: 9675185

Abstract

Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the ternary system ReLPS/DMPEd54/PLL(500) showed the features of a phase segregation phenomenon not affecting the transition temperatures of the pure lipid components, the ternary system composed of ReLPS/DMPEd54 and OmpF exhibited phase transition curves that were characterized by an unspecific (DMPEd54/OmpF) and a strong and unique (ReLPS/OmpF) type of lipid-protein interaction. Furthermore, semiquantitative estimations supported the supposition that OmpF might be able to induce bilayer asymmetry in preformed symmetrical ReLPS/DMPEd54 vesicles.

Full Text

The Full Text of this article is available as a PDF (343.4 KB).

Selected References

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

  1. Ames G. F., Spudich E. N., Nikaido H. Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J Bacteriol. 1974 Feb;117(2):406–416. doi: 10.1128/jb.117.2.406-416.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boggs J. M., Wood D. D., Moscarello M. A., Papahadjopoulos D. Lipid phase separation induced by a hydrophobic protein in phosphatidylserine--phosphatidylcholine vesicles. Biochemistry. 1977 May 31;16(11):2325–2329. doi: 10.1021/bi00630a003. [DOI] [PubMed] [Google Scholar]
  3. Casal H. L., Mantsch H. H. Polymorphic phase behaviour of phospholipid membranes studied by infrared spectroscopy. Biochim Biophys Acta. 1984 Dec 4;779(4):381–401. doi: 10.1016/0304-4157(84)90017-0. [DOI] [PubMed] [Google Scholar]
  4. Chapman D., Gómez-Fernández J. C., Goñi F. M. Intrinsic protein--lipid interactions. Physical and biochemical evidence. FEBS Lett. 1979 Feb 15;98(2):211–223. doi: 10.1016/0014-5793(79)80186-6. [DOI] [PubMed] [Google Scholar]
  5. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. Crystal structures explain functional properties of two E. coli porins. Nature. 1992 Aug 27;358(6389):727–733. doi: 10.1038/358727a0. [DOI] [PubMed] [Google Scholar]
  6. Curatolo W., Sakura J. D., Small D. M., Shipley G. G. Protein-lipid interactions: recombinants of the proteolipid apoprotein of myelin with dimyristoyllecithin. Biochemistry. 1977 May 31;16(11):2313–2319. doi: 10.1021/bi00630a001. [DOI] [PubMed] [Google Scholar]
  7. Dluhy R. A., Mendelsohn R., Casal H. L., Mantsch H. H. Interaction of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine-d54 mixtures with glycophorin. A fourier transform infrared investigation. Biochemistry. 1983 Mar 1;22(5):1170–1177. doi: 10.1021/bi00274a028. [DOI] [PubMed] [Google Scholar]
  8. Fried V. A., Rothfield L. I. Interactions between lipopolysaccharide and phosphatidylethanolamine in molecular monolayers. Biochim Biophys Acta. 1978 Dec 4;514(1):69–82. doi: 10.1016/0005-2736(78)90077-9. [DOI] [PubMed] [Google Scholar]
  9. Galanos C., Lüderitz O. Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur J Biochem. 1975 Jun;54(2):603–610. doi: 10.1111/j.1432-1033.1975.tb04172.x. [DOI] [PubMed] [Google Scholar]
  10. Garavito R. M., Rosenbusch J. P. Isolation and crystallization of bacterial porin. Methods Enzymol. 1986;125:309–328. doi: 10.1016/s0076-6879(86)25027-2. [DOI] [PubMed] [Google Scholar]
  11. Gomez-Fernandez J. C., Goni F. M., Bach D., Restall C. J., Chapman D. Protein-lipid interaction. Biophysical studies of (Ca2+ + Mg2+)-ATPase reconstituted systems. Biochim Biophys Acta. 1980 Jun 6;598(3):502–516. doi: 10.1016/0005-2736(80)90031-0. [DOI] [PubMed] [Google Scholar]
  12. Haverstick D. M., Glaser M. Influence of proteins on the reorganization of phospholipid bilayers into large domains. Biophys J. 1989 Apr;55(4):677–682. doi: 10.1016/S0006-3495(89)82866-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heyn M. P., Blume A., Rehorek M., Dencher N. A. Calorimetric and fluorescence depolarization studies on the lipid phase transition of bacteriorhodopsin--dimyristoylphosphatidylcholine vesicles. Biochemistry. 1981 Dec 8;20(25):7109–7115. doi: 10.1021/bi00528a009. [DOI] [PubMed] [Google Scholar]
  14. Jones N. C., Osborn M. J. Interaction of Salmonella typhimurium with phospholipid vesicles. Incorporation of exogenous lipids into intact cells. J Biol Chem. 1977 Oct 25;252(20):7398–7404. [PubMed] [Google Scholar]
  15. Kamio Y., Nikaido H. Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium. Biochemistry. 1976 Jun 15;15(12):2561–2570. doi: 10.1021/bi00657a012. [DOI] [PubMed] [Google Scholar]
  16. Kato N., Ohta M., Kido N., Ito H., Naito S., Hasegawa T., Watabe T., Sasaki K. Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli. J Bacteriol. 1990 Mar;172(3):1516–1528. doi: 10.1128/jb.172.3.1516-1528.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Katowsky M., Sabisch A., Gutberlet T., Bradaczek H. Molecular modelling of bacterial deep rough mutant lipopolysaccharide of Escherichia coli. Eur J Biochem. 1991 May 8;197(3):707–716. doi: 10.1111/j.1432-1033.1991.tb15962.x. [DOI] [PubMed] [Google Scholar]
  18. Kouaouci R., Silvius J. R., Graham I., Pézolet M. Calcium-induced lateral phase separations in phosphatidylcholine-phosphatidic acid mixtures. A Raman spectroscopic study. Biochemistry. 1985 Dec 3;24(25):7132–7140. doi: 10.1021/bi00346a017. [DOI] [PubMed] [Google Scholar]
  19. Labischinski H., Barnickel G., Bradaczek H., Naumann D., Rietschel E. T., Giesbrecht P. High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane. J Bacteriol. 1985 Apr;162(1):9–20. doi: 10.1128/jb.162.1.9-20.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mabrey S., Sturtevant J. M. Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3862–3866. doi: 10.1073/pnas.73.11.3862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marsh D., Watts A., Knowles P. F. Cooperativity of the phase transition in single- and multibilayer lipid vesicles. Biochim Biophys Acta. 1977 Mar 17;465(3):500–514. doi: 10.1016/0005-2736(77)90268-1. [DOI] [PubMed] [Google Scholar]
  22. Marvin H. J., ter Beest M. B., Hoekstra D., Witholt B. Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells. J Bacteriol. 1989 Oct;171(10):5268–5275. doi: 10.1128/jb.171.10.5268-5275.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nabedryk E., Garavito R. M., Breton J. The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation. Biophys J. 1988 May;53(5):671–676. doi: 10.1016/S0006-3495(88)83148-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Onishi H. R., Pelak B. A., Gerckens L. S., Silver L. L., Kahan F. M., Chen M. H., Patchett A. A., Galloway S. M., Hyland S. A., Anderson M. S. Antibacterial agents that inhibit lipid A biosynthesis. Science. 1996 Nov 8;274(5289):980–982. doi: 10.1126/science.274.5289.980. [DOI] [PubMed] [Google Scholar]
  26. Petersen N. O., Kroon P. A., Kainoshio M., Chan S. I. Thermal phase transitions in deuterated lecithin bilayers. Chem Phys Lipids. 1975 Aug;14(4):343–349. doi: 10.1016/0009-3084(75)90071-7. [DOI] [PubMed] [Google Scholar]
  27. Rietschel E. T., Brade H. Bacterial endotoxins. Sci Am. 1992 Aug;267(2):54–61. doi: 10.1038/scientificamerican0892-54. [DOI] [PubMed] [Google Scholar]
  28. Rietschel E. T., Kirikae T., Schade F. U., Mamat U., Schmidt G., Loppnow H., Ulmer A. J., Zähringer U., Seydel U., Di Padova F. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994 Feb;8(2):217–225. doi: 10.1096/fasebj.8.2.8119492. [DOI] [PubMed] [Google Scholar]
  29. Rothschild K. J., Clark N. A. Polarized infrared spectroscopy of oriented purple membrane. Biophys J. 1979 Mar;25(3):473–487. doi: 10.1016/S0006-3495(79)85317-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rottem S., Leive L. Effect of variations in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coli. J Biol Chem. 1977 Mar 25;252(6):2077–2081. [PubMed] [Google Scholar]
  31. Schletter J., Heine H., Ulmer A. J., Rietschel E. T. Molecular mechanisms of endotoxin activity. Arch Microbiol. 1995 Dec;164(6):383–389. doi: 10.1007/BF02529735. [DOI] [PubMed] [Google Scholar]
  32. Takeuchi Y., Nikaido H. Persistence of segregated phospholipid domains in phospholipid--lipopolysaccharide mixed bilayers: studies with spin-labeled phospholipids. Biochemistry. 1981 Feb 3;20(3):523–529. doi: 10.1021/bi00506a013. [DOI] [PubMed] [Google Scholar]
  33. Tardieu A., Luzzati V., Reman F. C. Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases. J Mol Biol. 1973 Apr 25;75(4):711–733. doi: 10.1016/0022-2836(73)90303-3. [DOI] [PubMed] [Google Scholar]
  34. Vaara M. Lipid A: target for antibacterial drugs. Science. 1996 Nov 8;274(5289):939–940. doi: 10.1126/science.274.5289.939. [DOI] [PubMed] [Google Scholar]
  35. Vaara M., Vaara T. Polycations as outer membrane-disorganizing agents. Antimicrob Agents Chemother. 1983 Jul;24(1):114–122. doi: 10.1128/aac.24.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vaara M., Vaara T. Polycations sensitize enteric bacteria to antibiotics. Antimicrob Agents Chemother. 1983 Jul;24(1):107–113. doi: 10.1128/aac.24.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang S., Martin E., Cimino J., Omann G., Glaser M. Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer. Biochemistry. 1988 Mar 22;27(6):2033–2039. doi: 10.1021/bi00406a033. [DOI] [PubMed] [Google Scholar]
  38. Wiese A., Schröder G., Brandenburg K., Hirsch A., Welte W., Seydel U. Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans. Biochim Biophys Acta. 1994 Mar 23;1190(2):231–242. doi: 10.1016/0005-2736(94)90079-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES