Lipid-protein interactions in Escherichia coli membranes over-expressing the sugar-H(+) symporter, GalP EPR of spin-labelled lipids.
The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.
Hubert
Anne
A
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Henderson
Peter J F
PJ
Marsh
Derek
D
D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013113:Spin Labels; D013696:Temperature
D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; D008563:Membrane Lipids; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D013113:Spin Labels; C023550:galactose-binding protein