Effects of lactose permease on the phospholipid environment in which it is reconstituted: a fluorescence and atomic force microscopy study.
The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.
Merino
Sandra
S
Departament de Fisicoquímica and Química Física, Universitat de Barcelona, E-08028 Barcelona, Spain.
Domènech
Oscar
O
Viñas
M
M
Montero
M Teresa
MT
Hernández-Borrell
Jordi
J
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005454:Fluorescence Polarization; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry*; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013499:Surface Properties
D008051:Lipid Bilayers; D008081:Liposomes; D026901:Membrane Transport Proteins; D010743:Phospholipids; C021499:lactose permease