Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

Interaction of a Homologous Series of Amphiphiles with P-glycoprotein in a Membrane Environment-Contributions of Polar and Non-Polar Interactions.

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [125I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P-gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P-gp's substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P-gp's binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes.

Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.

Effect of amphipathic HIV fusion inhibitor peptides on POPC and POPC/cholesterol membrane properties: a molecular simulation study.

T-20 and T-1249 fusion inhibitor peptides were shown to interact with 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC) (liquid disordered, ld) and POPC/cholesterol (1:1) (POPC/Chol) (liquid ordered, lo) bilayers, and they do so to different extents. Although they both possess a tryptophan-rich domain (TRD), T-20 lacks a pocket binding domain (PBD), which is present in T-1249. It has been postulated that the PBD domain enhances FI interaction with HIV gp41 protein and with model membranes. Interaction of these fusion inhibitor peptides with both the cell membrane and the viral envelope membrane is important for function, i.e., inhibition of the fusion process. We address this problem with a molecular dynamics approach focusing on lipid properties, trying to ascertain the consequences and the differences in the interaction of T-20 and T-1249 with ld and lo model membranes. T-20 and T-1249 interactions with model membranes are shown to have measurable and different effects on bilayer structural and dynamical parameters. T-1249's adsorption to the membrane surface has generally a stronger influence in the measured parameters. The presence of both binding domains in T-1249 appears to be paramount to its stronger interaction, and is shown to have a definite importance in membrane properties upon peptide adsorption.

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.

Molecular dynamics simulations of T-20 HIV fusion inhibitor interacting with model membranes.

T-20 (also known as enfuvirtide) is a fusion inhibitor peptide known to have some effectiveness in the control of progression of HIV infection by inhibiting the fusion of the HIV envelope with the target cell membrane. Recent results indicate that T-20 is able to interact with membranes in the liquid disordered state but not with membranes in an ordered state, which could be linked to its effectiveness. A detailed molecular picture of the interaction of these molecules with membranes is still lacking. To this effect, extensive molecular dynamics simulations (100 ns) were carried out to investigate the interaction between T-20 and bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and POPC/cholesterol (1:1). Membrane properties such as area/lipid, density profiles, order parameters and membrane thickness were studied. It was observed that T-20 has the ability to interact to different extents with both model membranes in this study and that peptide interaction with the bilayer surface has a local effect on membrane structure. The formation of hydrogen bonding between certain peptide residues and the POPC phosphate group was observed. However, T-20 showed a more limited extent of interaction with model membranes when compared with other, more efficient, peptides (such as T-1249). This effect is most notable in POPC/Chol membranes in which interaction is especially weak, owing to less peptide residues acting as H bond donors to POPC and virtually no H bonds being formed between T-20 and cholesterol. This lower ability to interact with membranes is probably correlated with its smaller inhibitory efficiency.

Interaction of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled fatty amines with 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine bilayers: a molecular dynamics study.

A complete homologous series of fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled fatty amines of varying alkyl chain length, NBD-C(n), inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, was studied using atomistic molecular dynamics (MD) simulations. For all amphiphiles, the NBD fluorophore locates near the glycerol backbone/carbonyl region of POPC and establishes stable hydrogen bonding with POPC ester oxygen atoms. Small differences observed in the transverse location of the fluorophore correlate with other calculated parameters and with small discrepancies recently measured in the photophysical properties of the molecules. The longer-chained NBD-C(n) amphiphiles show significant mass density near the bilayer midplane, and the chains of these derivatives interdigitate to some extent the opposite bilayer leaflet. This phenomenon leads to a slower lateral diffusion for the longer-chained derivatives (n > 12). Effects of these amphiphiles on the structure and dynamics of the host lipid were found to be relatively mild, in comparison with acyl-chain-labeled NBD probes. The molecular details obtained by this work allow the rationalization of the nonmonotonic behavior, recently obtained experimentally, for the photophysical parameters of the amphiphiles and the kinetic and thermodynamic parameters for their interaction with the POPC membranes.

Characterization of peptide-induced morphological alterations in membranes by fluorescence resonance energy transfer.

Interaction of fusogenic or membrane-perturbing peptides with lipid bilayers often involves drastic rearrangements of the membrane structure, with redistribution of lipids, inducement of bilayer curvature, or formation of non-bilayer or multibilayer structures. Fluorescence (or Förster) Resonance Energy Transfer (FRET) is a photophysical technique which has an acute sensitivity to distances in the nanometer range, and, as such, is particularly suited to probe alterations in membrane organization in this length scale. This article reviews methods and selected applications of FRET in this field, from the now classic (fusion induced) lipid-mixing assay to examples where kinetic modeling of FRET enables the recovery of topological information.

Sifuvirtide screens rigid membrane surfaces. establishment of a correlation between efficacy and membrane domain selectivity among HIV fusion inhibitor peptides.

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion inhibitor, presently in clinical trials. Because of the aromatic amino acid residues of the peptide, its behavior in aqueous solution and the interaction with lipid-membrane model systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy techniques (both steady-state and time-resolved). No significant aggregation of the peptide was observed with aqueous solution. Various biological and nonbiological lipid-membrane compositions were analyzed, and atomic force microscopy was used to visualize phase separation in several of those mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquid-ordered phase). However, significant partitioning was observed with the positively charged lipid models (K(p) = (2.2 +/- 0.3) x 10(3)), serving as a positive control. Fluorescence quenching using Förster resonance acrylamide and lipophilic probes was carried out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at the surface of these membranes was observed and confirmed by using Förster resonance energy-transfer experiments. These results indicate a targeting of the peptide to gel-phase domains relatively to liquid-disordered or liquid-ordered phase domains. This larger affinity and selectivity toward the more rigid areas of the membranes, where most of the receptors are found, or to viral membrane, may help explain the improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the peptide at the fusion site.

Structure and dynamics of the gammaM4 transmembrane domain of the acetylcholine receptor in lipid bilayers: insights into receptor assembly and function.

A 28-mer peptide (gammaM4) corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor (AChR) gamma-subunit, with a single tryptophan residue (Trp6), was reconstituted into lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), loaded with either high or low amounts of cholesterol, i.e., in the conjugated liquid-ordered and liquid-disordered phases, respectively, at room temperature. By making use of the Trp intrinsic fluorescence, both steady-state and time-resolved fluorescence techniques were employed, namely, red-edge excitation shift effect, decay-associated spectra (DAS), and time-resolved anisotropy. The results obtained here, together with previous studies on the same reconstituted peptide, indicate that: (i) Trp6 is strongly anchored in the bilayer with a defined transverse location; (ii) the modifications in the measured DAS are related to the complex result of a self-quenching process on the decay parameters; (iii) the wobbling movement of the indole moiety of Trp6 is fast but severely restricted in amplitude; and, (iv) in the liquid-ordered phase, the bilayer properties and the tilt angle of the peptide enhance peptide-peptide interactions, with the formation of peptide rich patches and possibly some anti-parallel helix-helix aggregates, showing different dynamics from that of the peptide in the liquid-disordered phase where the peptide is randomly distributed.

Structural effects of a basic peptide on the organization of dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine membranes: a fluorescent resonance energy transfer study.

We studied the effect of a model basic peptide, hexalysiltryptophan, on the organization of dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine unilamellar vesicles by means of fluorescent resonance energy transfer (FRET) between fluorescently labeled phospholipids. Several FRET theoretical models assuming different bilayer geometries and probe distributions were fitted to the time-resolved data. The experiments were carried out at two temperatures in different regions of the lipid mixture phase diagram. At 45 degrees C, the expected gel/fluid phase separation was verified by model fitting in peptide-free vesicles, which from the FRET approach means that domains are larger than approximately 200 A. No noticeable alteration of membrane organization was detected upon increasing the peptide concentration. At variance, for the single fluid phase at 60 degrees C, there was a large increase in FRET efficiency upon peptide addition to the lipid vesicles, mainly caused by peptide-induced vesicle aggregation. The system gradually changed from unilamellar lipid vesicles to a multibilayer geometry, and a limit lamellar repeat distance of approximately 57 A was recovered. Furthermore, no evidence for lateral domain formation on the FRET length scale was found at this temperature, the cationic peptide being only able to induce local lipid demixing, causing a short-range sequestration of 2-3 acidic lipids around each surface-adsorbed peptide.

Cholesterol modulates the organization of the gammaM4 transmembrane domain of the muscle nicotinic acetylcholine receptor.

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Förster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.

Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies.

The application of fluorescence methodologies to obtain information about the extent, dynamics and topology of peptide interaction with binary phospholipid (mainly zwitterionic/anionic) mixtures is reviewed. First, general approaches based on peptide (tryptophan residues) fluorescence properties that give information about its partition, location and dynamics will be presented. Then, methodologies based on membrane probes fluorescence that report the influence of peptide binding and/or incorporation on the lateral organization (phase separation) of membrane phospholipids will be described. Specific examples taken from the literature that illustrate both situations are presented as well as formalisms for data analysis. It is shown that steady-state and time-resolved fluorescence data (particularly important in the case of fluorescence resonance energy transfer studies) give complementary information, allowing a molecular picture of peptide interaction with biphasic systems to be drawn.