The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.
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.
Robert Koch-Institut, 13353 Berlin, Germany.
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D001426:Bacterial Proteins / Q000737:chemistry; D003903:Deuterium; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D004926:Escherichia coli; D008070:Lipopolysaccharides / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008962:Models, Theoretical; D008968:Molecular Conformation; D009154:Mutation; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D011107:Polylysine / Q000737:chemistry*; D018272:Porins / Q000737:chemistry*; D011487:Protein Conformation; D012475:Salmonella / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D008081:Liposomes; C109237:OmpF protein; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D018272:Porins; D011107:Polylysine; D003903:Deuterium; D004134:Dimyristoylphosphatidylcholine; C018525:1,2-dimyristoylphosphatidylethanolamine