Effect of lactose permease presence on the structure and nanomechanics of two-component supported lipid bilayers.

In this paper we present a comparative study of supported lipid bilayers (SLBs) and proteolipid sheets (PLSs) obtained from deposition of lactose permease (LacY) of Escherichia coli proteoliposomes in plane. Lipid matrices of two components, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), at a 3:1, mol/mol ratio, were selected to mimic the inner membrane of the bacteria. The aim was to investigate how species of different compactness and stiffness affect the integration, distribution and nanomechanical properties of LacY in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). Both compositions displayed phase separation and were investigated by atomic force microscopy (AFM) imaging and force-spectroscopy (FS) mode. PLSs displayed two separated, segregated domains with different features that were characterised by FS and force-volume mode. We correlated the nanomechanical characteristics of solid-like gel phase (Lß) and fluid liquid-crystalline phase (Lα) with phases emerging in presence of LacY. We observed that for both compositions, the extended PLSs showed a Lß apparently formed only by lipids, whilst the second domain was enriched in LacY. The influence of the lipid environment on LacY organisation was studied by performing protein unfolding experiments using the AFM tip. Although the pulling experiments were unspecific, positive events were obtained, indicating the influence of the lipid environment when pulling the protein. A possible influence of the lateral surface pressure on this behaviour is suggested by the higher force required to pull LacY from DPPE:POPG than from POPE:POPG matrices. This is related to higher forces governing protein-lipid interaction in presence of DPPE.

Modeling FRET to investigate the selectivity of lactose permease of Escherichia coli for lipids.

Förster resonance energy transfer (FRET) is a photophysical process by which a donor (D) molecule in an electronic excited state transfers its excitation energy to a second species, the acceptor (A). Since FRET efficiency depends on D-A separation, the measurement of donor fluorescence in presence and absence of the acceptor allows determination of this distance, and therefore FRET has been extensively used as a "spectroscopic ruler". In membranes, interpretation of FRET is more complex, since one D may be surrounded by many A molecules. Such is the case encountered with membrane proteins and lipids in the bilayer. This paper reviews the application of a model built to analyze FRET data between a single tryptophan mutant of the transmembrane protein lactose permease (W151/C154G of LacY), the sugar/H(+) symporter from Escherichia coli, and different pyrene-labeled phospholipids. Several variables of the system with biological implication have been investigated: The selectivity of LacY for different species of phospholipids, the enhancement of the sensitivity of the FRET modeling, and the mutation of a particular aminoacid (D68C) of the protein. The results obtained support: (i) Preference of LacY for phosphatidylethanolamine (PE) over phosphatidylglycerol (PG); (ii) affinity of LacY for fluid (L(α)) phases; and (iii) importance of the aspartic acid in position 68 in the sequence of LacY regarding the interaction with the phospholipid environment. Besides, by exploring the enhancement of the sensitivity by using pure lipid matrices with higher mole fractions of labelled-phospholipid, the dependence on acyl chain composition is unveiled.

Miscibility behavior and nanostructure of monolayers of the main phospholipids of Escherichia coli inner membrane.

We report a thermodynamic study of the effect of calcium on the mixing properties at the air-water interface of two phospholipids that mimic the inner membrane of Escherichia coli: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. In this study, pure POPE and POPG monolayers and three mixed monolayers, χ(POPE) = 0.25, 0.5, and 0.75, were analyzed. We show that for χ(POPE) = 0.75, the values of the Gibbs energy of mixing were negative, which implies attractive interactions. We used atomic force microscopy to study the structural properties of Langmuir-Blodgett monolayers that were transferred onto mica substrate at lateral surface pressures of 25 and 30 mN m(-1). The topographic images of pure POPE and POPG monolayers exhibited two domains of differing size and morphology, showing a step height difference within the range expected for liquid-condensed and liquid-expanded phases. The images captured for χ(POPE) = 0.25 were featureless, and for χ(POPE) = 0.5 small microdomains were observed. The composition that mimics quantitatively the proportions found in the inner membrane of E. coli , χ(POPE) = 0.75, showed large liquid condensed domains in the liquid expanded phase. The extension of each domain was quantitatively analyzed. Because calcium is used in the formation of supported bilayers of negatively charged phospholipids, the possible influence of the nanostructure of the apical on the distal monolayer is discussed.

Lactose permease lipid selectivity using Förster resonance energy transfer.

The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better this in the present work we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labeled phospholipids (Pyr-PE and Pyr-PG) at 37 degrees C. We have reconstituted this LacY mutant in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE/PG proportion found in the E. coli inner membrane (3:1, mol/mol). A theoretical model has been fitted to the experimental data. In the POPE/POPG system, quantitative model calculations show accordance with the experimental values that requires an annular region composed of approximately approximately 90 mol% PE. The experimental FRET efficiencies for the gel/fluid phase-separated DOPE/POPG system indicate a higher presence of PG in the annular region, from which it can be concluded that LacY shows clear preference for the fluid phase. Similar conclusions are obtained from analysis of excimer-to-monomer (E/M) pyrene ratios. To test the effects of this on cardiolipin (CL) on the annular region, myristoyl-CL and oleoyl-CL were incorporated in the biomimetic POPE/POPG matrix. The experimental FRET efficiency values, slightly larger for Pyr-PE than for Pyr-PG, suggest that CL displaces POPE and, more extensively, POPG from the annular region of LacY. Model fitting indicates that CL enrichment in the annular layer is, in fact, solely produced by replacing PG and that myristoyl-CL is not able to displace PE in the same way that oleoyl-CL does. One of the conclusions of this work is the fact that LacY inserts preferentially in fluid phases of membranes.

Phospholipid-lactose permease interaction as reported by a head-labeled pyrene phosphatidylethanolamine: a FRET study.

Förster resonance energy transfer (FRET) measurements were performed in preceding works to study the selectivity between a single-tryptophan mutant of lactose permease (LacY) of Escherichia coli (used as the donor) and phospholipid probes labeled with pyrene at the acyl chain moiety (used as the acceptor). In the present work, we report the results obtained by using the same LacY mutant (W151/C154G) and binary lipid mixtures of phosphatidylethanolamine (PE) differing in the acyl chain composition and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (3:1 mol/mol) doped with a phospholipid probe labeled with pyrene at the headgroup. The use of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(1-pyrenesulfonyl) ammonium salt (HPyr-PE), which bears two unsaturated acyl chains, enabled the investigation of the specific interaction between LacY and HPyr-PE. The main conclusions raised from our results suggest that (i) for phase-separated systems, LacY would be located in fluid domains nominally enriched in POPG, and if a given proportion of PE is present in this phase, it will be mainly located around LacY; and (ii) in the absence of phase separation, LacY is preferentially surrounded by PE and, in particular, seems to be sensitive to the lipid spontaneous curvature.

Phosphatidylethanolamine-lactose permease interaction: a comparative study based on FRET.

In this work we have investigated the selectivity of lactose permease (LacY) of Escherichia coli (E. coli) for its surrounding phospholipids when reconstituted in binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). Förster resonance energy transfer (FRET) measurements have been performed to investigate the selectivity between a single tryptophan mutant of LacY used as donor (D), and two analogues of POPE and POPG labeled with pyrene in the acyl chains (Pyr-PE and Pyr-PG) used as acceptors. As a difference from previous works, now the donor has been single-W151/C154G/D68C LacY. It has been reported that the replacement of the aspartic acid in position 68 by cysteine inhibits active transport in LacY. The objectives of this work were to elucidate the phospholipid composition of the annular region of this mutant and to determine whether the mutation performed, D68C, induced changes in the protein-lipid selectivity. FRET efficiencies for Pyr-PE were always higher than for Pyr-PG. The values of the probability of each site in the annular ring being occupied by a label (µ) were similar at the studied temperatures (24 °C and 37 °C), suggesting that the lipid environment is not significantly affected when increasing the temperature. By comparing the results with those obtained for single-W151/C154G LacY, we observe that the mutation in the 68 residue indeed changes the selectivity of the protein for the phospholipids. This might be probably due to a change in the conformational dynamics of LacY.

Membrane protein-lipid selectivity: enhancing sensitivity for modeling FRET data.

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.

Acyl chain differences in phosphatidylethanolamine determine domain formation and LacY distribution in biomimetic model membranes.

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the two main components of the inner membrane of Escherichia coli. It is well-known that inner membrane contains phospholipids with a nearly constant polar headgroup composition. However, bacteria can regulate the degree of unsaturation of the acyl chains in order to adapt to different external stimuli. Studies on model membranes of mixtures of PE and PG, mimicking the proportions found in E. coli, can provide essential information on the phospholipid organization in biological membranes and may help in the understanding of membrane proteins activity, such as lactose permease (LacY) of E. coli. In this work we have studied how different phosphatidylethanolamines differing in acyl chain saturation influence the formation of laterally segregated domains. Three different phospholipid systems were studied: DOPE:POPG, POPE:POPG, and DPPE:POPG at molar ratios of 3:1. Lipid mixtures were analyzed at 24 and 37 °C through three different model membranes: monolayers, liposomes, and supported lipid bilayers (SLBs). Data from three different techniques, Langmuir isotherms, Laurdan generalized polarization, and atomic force microscopy (AFM), evidenced that only the DPPE:POPG system exhibited coexistence between gel (L(ß)) and fluid (L(α)) phases at both 24 and 37 °C . In the POPE:POPG system the L(ß)/L(α) coexistence appears at 27 °C. Therefore, in order to investigate the distribution of LacY among phospholipid phases, we have used AFM to explore the distribution of LacY in SLBs of the three phospholipid systems at 27 °C, where the DOPE:POPG is in L(α) phase and POPE:POPG and DPPE:POPG exhibit L(ß)/L(α) coexistence. The results demonstrate the preferential insertion of LacY in fluid phase.

Force spectroscopy study of Langmuir-Blodgett asymmetric bilayers of phosphatidylethanolamine and phosphatidylglycerol.

Phosphatidylethanolamine (PE) and phosphatidylgycerol (PG) are the main components of the inner membrane of Escherichia coli. Mixtures of PE and PG mimicking the proportions found in E. coli have been extensively used to reconstitute transmembrane proteins as lactose permease (LacY) in proteoliposomes because in this environment the protein shows maximal activity. Hence, the study of the physicochemical properties of this phospholipid matrix becomes of potential interest. In previous studies, we used atomic force microscopy (AFM) and force spectroscopy (FS) to study the topographic and nanomechanical properties of supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and of POPE and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). The study reported here was extended for completeness to asymmetric SLBs obtained by the Langmuir-Blodgett (LB) method. Thus, we prepared SLBs with the proximal leaflet extracted at 30 mN x m(-1) and the distal leaflet extracted at 25 mN x m(-1). We prepared SLBs with both leaflets with same composition (POPG/POPG), and also with the proximal leaflet of POPE and the distal leaflet of POPG or POPE:POPG (3:1, mol/mol). The topography of the SLBs acquired in liquid was compared with the topography of the monolayers acquired in air. Breakthrough (F(y)) and adhesion forces (F(adh)) of SLBs were extracted from force curves. The values obtained are discussed in terms of the possible involvement of the nanomechanical properties of the SLBs in membrane protein insertion. The results provide means for the observation that insertion of LacY in POPE:POPG (3:1, mol/mol) occurs preferentially in the fluid phase, which is the phase with the lower F(y) and the higher F(adh).