Effects of lactose permease of Escherichia coli on the anisotropy and electrostatic surface potential of liposomes.
The membrane transport protein lactose permease (LacY), a member of the Major Facilitator Superfamily (MFS) containing twelve membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes of: (i) 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in equimolar proportions; and (ii) Escherichia coli total lipid extract. The structural order of the lipid membranes, in the presence and absence of LacY, was investigated using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) evidenced: (i) the insertion of LacY into the bilayer; and (ii) a surface effect on the membranes. The most dramatic effects were observed when LacY was reconstituted in the E. coli lipid matrix. The effect of the protein on the electrostatic surface potential of each bilayer was also examined using a fluorescent pH indicator, 4-Heptadecyl-7-hydroxycoumarin (HHC). Changes in surface potential were enhanced in the presence of the substrate (i.e. lactose) only when the lipid matrices were charged. These results suggest a role for charged phospholipids (i.e. phosphatidylethanolamine or phosphatidylglycerol) in proton transfer to the amino acids involved in substrate translocation.
Merino-Montero
Sandra
S
Departament de Fisicoquímica, Universitat de Barcelona, 08028- Barcelona, Spain.
Montero
M Teresa
MT
Hernández-Borrell
Jordi
J
D016880:Anisotropy; D001557:Benzenesulfonates / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004161:Diphenylhexatriene / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D055672:Static Electricity; D013696:Temperature
D001557:Benzenesulfonates; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D026901:Membrane Transport Proteins; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D004161:Diphenylhexatriene; C483858:phosphatidylethanolamine; C021499:lactose permease; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine