Membrane Protein–Lipid Selectivity: Enhancing Sensitivity for Modeling FRET Data

Abstract

Förster resonance energy transfer (FRET) is a powerful method for the characterization of membrane proteins lipid selectivity. FRET can be used to quantify distances between a single donor and a single acceptor molecule; however, for FRET donors and acceptors scattered in the bilayer plane, multiple donor-acceptor pairs and distances are present. In addition, when studying protein/lipid selectivity, for a single tryptophan used as a donor; several lipid acceptors may be located at the boundary region (annular lipids) of the protein. Therefore, in these experiments, a theoretical analysis based on binomial distribution of multiple acceptors around the membrane proteins is required. In this work, we performed FRET measurements between single tryptophan lactose permease (W151/C154G LacY) of Escherichia coli and pyrene-labeled phospholipids (Pyr-PE, Pyr-PG, and Pyr-PC) reconstituted in palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline, and 1,2-dioleoyl-sn-glycero-3-phospho-choline at 25 and 37 °C. To increase the sensitivity of the method and to ascertain the lipid selectivity for LacY, we reconstituted the protein in the pure phospholipids doped with 1.5% of labeled phospholipids. From fitting the theoretical model to the experimental FRET efficiencies, two parameters were calculated: the probability of a site in the annular ring being occupied by a labeled pyrene phospholipid and the relative association constant between the labeled and unlabeled phospholipids. The experimental FRET efficiencies have been interpreted taking into account the particular folding of the protein in each phospholipid matrix. Additional information on the annular lipid composition for each system has been obtained by exciting W151/C154G LacY and monitoring the emission intensities for monomer and excimer of the pyrene spectra. The results obtained indicate a higher selectivity of LacY for PE over PG and PC and pointed to a definite role of the acyl chains in the overall phospholipid-protein interaction.

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

“…However, proper folding may also be influenced by the properties of the annular lipid shell surrounding and in close association with membrane proteins. POPE was much more effective than DOPE in disrupting the interaction of PG with LacY in proteoliposomes, as measured by FRET between Trp-151 of LacY and pyrene-labeled PG (47). Therefore, the preference of LacY for POPE and POPC rather than DOPE and DOPC that we observed may be explained by the capacity of hetero-fatty acid phospholipids to exclude non-supportive anionic lipids from the annular shell surrounding LacY.…”

Phosphatidylethanolamine–Lactose Permease Interaction: A Comparative Study Based on FRET

“…4,15 This may be a crucial event that triggers the whole mechanism of active transport. Nevertheless, not only the headgroup is important: 26,27 in recent works where the protein was reconstituted in single phospholipid matrixes 21 and in others based on measuring transport of genetically modified bacteria, 28 the importance of the acyl chain moiety in the interaction between phospholipids and membrane proteins has been pointed out.…”

“…Afterward, large unilamellar liposomes (LUVs) supplemented with 0.2% of DDM were incubated overnight at room temperature. Liposomes were subsequently mixed with the solubilized protein and incubated at 4 °C for 30 20,21 based on the protection of the substrate against thiol modification of LacY. Briefly, 50 μL of proteoliposomes containing 1.5 μM single-W151/C154G/D68C LacY 22 were incubated at room temperature for 5 min with either 15 mM β-D-galactopyranosyl-1-thioβ-D-galactopyranoside (TDG) or 15 mM L-glucose.…”

“…The description of the model used to fit the data has been clarified in detail elsewhere. 21 Briefly, we assume the existence of two populations of acceptors (A), one located at the annular shell around the protein and another in the bulk outside it. Then we can write the decay of the fluorescence of the donor D as…”

“…In previous works − we have exploited Förster resonance energy transfer (FRET) to study lipid selectivity between a single tryptophan mutant of LacY (W151/C154G), used as donor (D), and different phospholipids labeled with pyrene in the acyl chains that are used as acceptors (A). In the present work we have used W151/C154G and delineated a new mutant W151/C154G/D68C, in which the aspartic acid residue has been replaced by cysteine.…”