Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of interaction between human serum albumin and N6-(2-hydroxyethyl)-adenosine by fluorescence spectroscopy and molecular modelling.

The interaction between human serum albumin (HSA) and N(6)-(2-hydroxyethyl)-adenosine (HEA) was investigated using fluorescence spectroscopy in combination with UV absorption spectroscopy for the first time. The results of spectroscopic measurements suggested that the hydrophobic interaction was the predominant intermolecular force stabilizing the complex, which was in good agreement with the results of molecular modelling study. The enthalpy change (DeltaH) and the entropy change (DeltaS) were calculated, according to the Van't Hoff equation, to be -24.05 kJ/mol and 30.23 J/mol/K, respectively. The effects of common ions on the binding constant of the HEA-HSA complex at room temperature were also investigated.     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Fluorescence quenching in lipid micelles and bilayers: Evaluation of bimolecular rate constants

Dynamic quenching of fluorophores and quenchers in lipid micelles and bilayers can yield information about the bimolecular rate constant for the quenching reaction, and hence information about the microviscosity of the fluorophore-quencher environment. When the fluorophore and quencher have relatively fixed transverse positions in the bilayer, the analysis of Sikaris et al. (Chem. Phys. Lipids. 29 (1981) 23) can be used to separate the microviscosity and proximity contributions to quenching. We now extend this method to show explicitly the effect of static quenching on the analysis. We show by simulation and experiment that a correction factor must be included when static quenching contributes to the observed quenching efficiency.     

The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP_1-19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP_1-19 in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP_1-19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP_1-19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.     

Dynamics of bolaamphiphilic fluorescent polyenes in lipid bilayers from polarization emission spectroscopy

The rotational motions of the biamphiphilic polyenes (bolapolyenes) dimethyl all-(E)-octacosa-10,12,14,16,18-pentaenedioate (DE28:5) and dimethyl all-(E)-tetratriaconta-13,15,17,19,21-pentaenedioate (DE34:5), with head-to-head distances of 34 and 42A, respectively, have been examined by fluorescence anisotropy methods. The membrane-spanning bolapolyenes, which contain a central emitting pentaene group tethered to two methoxycarbonyl opposite polar heads by symmetric C(8) (DE28:5) and C(11) (DE34:5) polymethylene chains, were dispersed in lipid bilayers of DPPC or DMPC, and the stationary and picosecond-resolved emission was recorded as a function of temperature. In fluid-phase DMPC bilayers, three relaxation times could be determined, assigned to fast (0.2 and 2ns) single-bond isomerization processes localized on the alkyl chains, and to whole-molecule oscillations ( approximately 11ns), respectively. The anisotropy decay parameters were further analyzed in terms of a diffusive model for wobbling in a Gaussian ordering potential, to assess the anchoring effect of the symmetric polar heads. In this way, the average rotational diffusion constant of the bolapolyenes, D( perpendicular), could be estimated as 0.022-0.026rad(2) ns(-1) (DMPC bilayers, 35 degrees Celsius), a value that is only 1/3 of that corresponding to the related pentaene fatty acid spanning a single membrane monolayer. In contrast, the amplitude of the equilibrium orientational distribution (theta(half-cone) approximately 50 degrees ) is very similar for both the transmembrane and the single-headed polyenes. The reorientational oscillations of the central emitting group in the bolapolyenes necessarily would produce large-amplitude (2-5A) and very fast (ns) translational motions of the polar heads.     

Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Nisin adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions and antibacterial activity

Study of peptides adsorption on surfaces remains a current challenge in literature. A complementary approach, combining X‐ray photoelectron spectroscopy (XPS) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) was used to investigate the antimicrobial peptide nisin adsorption on hydrophilic and hydrophobic surfaces. The native low density polyethylene was used as hydrophobic support and it was grafted with acrylic acid to render it hydrophilic. XPS permitted to confirm nisin adsorption and to determine its amount on the surfaces. ToF‐SIMS permitted to identify the adsorbed bacteriocin type and to observe its distribution and orientation behavior on both types of surfaces. Nisin was more oriented by its hydrophobic side to the hydrophobic substrate and by its hydrophilic side to the outer layers of the adsorbed peptide, in contrast to what was observed on the hydrophilic substrate. A correlation was found between XPS and ToF‐SIMS results, the types of interactions on both surfaces and the observed antibacterial activity. Such interfacial studies are crucial for better understanding the peptides interactions and adsorption on surfaces and must be considered when setting up antimicrobial surfaces. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetics and thermodynamics of association of a phospholipid derivative with lipid bilayers in liquid-disordered and liquid-ordered phases

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) in liquid-disordered and liquid-ordered phase lipid bilayer membranes, of the fluorescent phospholipid derivative NBD-dimyristoylphosphatidyl ethanolamine. This study made use of a recently described method that exploits a detailed knowledge of the binding kinetics of an amphiphile to bovine serum albumin, to recover the insertion and desorption rate constants when the albumin-bound amphiphile is transferred through the aqueous phase to the membrane and vice versa. The lipid bilayers, studied as large unilamellar vesicles, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine in the liquid-disordered phase; and from two cholesterol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and cholesterol (molar ratio of 1:1), and egg sphingomyelin and cholesterol (molar ratio of 6:4), both in the liquid-ordered phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were directly measured between 15 degrees and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes, were derived from this data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and permitted the estimation of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane.     

Antimicrobial peptides temporins B and L induce formation of tubular lipid protrusions from supported phospholipid bilayers

The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.     

Effects of lithium on the human erythrocyte membrane and molecular models

The mechanism whereby lithium carbonate controls manic episodes and possibly influences affective disorders is not yet known. There is evidence, however, that lithium alters sodium transport and may interfere with ion exchange mechanisms and nerve conduction. For these reasons it was thought of interest to study its perturbing effects upon membrane structures. The effects of lithium carbonate (Li+) on the human erythrocyte membrane and molecular models have been investigated. The molecular models consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. This report presents the following evidence that Li+ interacts with cell membranes: a) X-ray diffraction indicated that Li+ induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC and DMPE; b) experiments performed on DMPC large unilamellar vesicles (LUV) by fluorescence spectroscopy also showed that Li+ interacted with the lipid polar groups and hydrophobic acyl chains, and c) in scanning electron microscopy (SEM) studies on intact human erythrocytes the formation of echinocytes was observed, effect that might be due to the insertion of Li+ in the outer monolayer of the red cell membrane.     

Monomethylarsonate (MMAv) exerts stronger effects than arsenate on the structure and thermotropic properties of phospholipids bilayers

Methylation of inorganic arsenic has been regarded as a detoxification mechanism because its metabolites monomethylarsonic acid (MMA(v)) and dimethylarsinic acid (DMA(v)) are supposed to be less toxic than inorganic arsenite and arsenate. In recent years, however, this interpretation has been questioned. Additionally, there are insufficient reports concerning the effects of arsenic compounds on cell membrane structure and functions. With the aim to better understand the molecular mechanisms of the interaction of MMA(v) and arsenate with cell membranes, we have utilized molecular models consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of many cell membranes including that of the human erythrocyte. The capacity of MMA(v) and arsenate to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction; the modifications of their thermotropic behavior were followed by differential scanning calorimetry (DSC), while DMPC large unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. It was found that MMA(v) and arsenate did not structurally perturb DMPC bilayers; however, DMPE bilayers did suffer structural perturbations by MMA(v). DSC measurements also revealed that DMPE's thermotropic properties were significantly affected by arsenicals, where MMA(v) was more effective than arsenate, whilst only slight modifications were observed in the case of DMPC-MMA(v) system.     

Structural effects in vitro of the anti-inflammatory drug diclofenac on human erythrocytes and molecular models of cell membranes

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly on those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated and reported. This report presents the following evidence that diclofenac interacts with red cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that diclofenac interacted with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes it was observed that the drug induced changes different from the normal biconcave morphology of most red blood cells. This is the first time in which structural effects of diclofenac on the human erythrocyte membrane have been described.     

Kinetics and thermodynamics of association of a fluorescent lysophospholipid derivative with lipid bilayers in liquid-ordered and liquid-disordered phases

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) of the fluorescent lysophospholipid derivative NBD-lyso-1-myristoylphosphatidylethanolamine in l(d) and l(o) phase lipid bilayer membranes. The lipid bilayers, studied as LUV, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine, in the l(d) phase; and from two Chol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and Chol (molar ratio of 1:1) and SpM and Chol (molar ratio of 6:4), both in the l(o) phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were measured between 15 degrees C and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes were derived from these data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and an estimation was made of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane. We show that, contrary to general belief, the association of NBD-lysoMPE with lipid bilayers is not a diffusion-controlled process, the rate-limiting step in insertion being the formation of a free area in the membrane surface of an adequate size for insertion to occur.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

Transbilayer movement of phospholipids at the main phase transition of lipid membranes: implications for rapid flip-flop in biological membranes

The transbilayer movement of fluorescent phospholipid analogs in liposomes was studied at the lipid phase transition of phospholipid membranes. Two NBD-labeled analogs were used, one bearing the fluorescent moiety at a short fatty acid chain in the sn-2 position (C(6)-NBD-PC) and one headgroup-labeled analog having two long fatty acyl chains (N-NBD-PE). The transbilayer redistribution of the analogs was assessed by a dithionite-based assay. We observed a drastic increase of the transbilayer movement of both analogs at the lipid phase transition of DPPC (T(c) = 41 degrees C) and DMPC (T(c) = 23 degrees C). The flip-flop of analogs was fast at the T(c) of DPPC with a half-time (t(1/2)) of ~6-10 min and even faster at the T(c) of DMPC with t(1/2) on the order of <2 min, as shown for C(6)-NBD-PC. Suppressing the phase transition by the addition of cholesterol, the rapid transbilayer movement was abolished. Molecular packing defects at the phase transition are assumed to be responsible for the rapid transbilayer movement. The relevance of those defects for understanding of the activity of flippases is discussed.