Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

Binding of imipramine to phospholipid bilayers using radioligand binding assay

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.     

Mechanism of membrane binding by the bovine seminal plasma protein,PDC-109: a surface plasmon resonance study

PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k(-1)) for the DMPC/PDC-109 system was found to be 2.7 x 10(-2) s(-1) whereas the k(-1) values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10(7) M(-1). The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.     

Effects of calcium-induced aggregation on the physical stability of liposomes containing plant glycolipids

Membranes containing either negatively charged lipids or glycolipids can be aggregated by millimolar concentrations of Ca(2+). In the case of membranes made from the negatively charged phospholipid phosphatidylserine, aggregation leads to vesicle fusion and leakage. However, some glycolipid-containing biological membranes such as plant chloroplast thylakoid membranes naturally occur in an aggregated state. In the present contribution, the effect of Ca(2+)-induced aggregation on membrane stability during freezing and in highly concentrated salt solutions (NaCl+/-CaCl(2)) has been determined in membranes containing different fractions of uncharged galactolipids, or a negatively charged sulfoglucolipid, or the negatively charged phospholipid phosphatidylglycerol (PG), in membranes made from the uncharged phospholipid phosphatidylcholine (PC). In the case of the glycolipids, aggregation did not lead to fusion or leakage even under stress conditions, while it did lead to fusion and leakage in PG-containing liposomes. Liposomes made from a mixture of glycolipids and PG that approximates the lipid composition of thylakoids were very unstable, both during freezing and at high solute concentrations and leakage and fusion were increased in the presence of Ca(2+). Collectively, the data indicate that the effects of Ca(2+)-induced aggregation of liposomes on membrane stability depend critically on the type of lipid involved in aggregation. While liposomes aggregated through glycolipids are highly stable, those aggregated through negatively charged lipids are severely destabilized.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and 'gas-dissected' membranes

The investigation focuses on the phospholipid composition of the sarcolemma of cultured neonatal rat heart cells and on the distribution of the phospholipid classes between the two monolayers of the sarcolemma. The plasma membranes are isolated by 'gas-dissection' technique and 38% of total cellular phospholipid is present in the sarcolemma with the composition: phosphatidylethanolamine (PE) 24.9%, phosphatidylcholine (PC) 52.0%, phosphatidylserine/phosphatidylinositol (PS/PI) 7.2%, sphingomyelin 13.5%. The cholesterol/phospholipid ratio of the sarcolemma is 0.5. The distribution of the phospholipids between inner and outer monolayer is defined with the use of two phospholipases A2, sphingomyelinase C or trinitrobenzene sulfonic acid as lipid membrane probes in whole cells. The probes have access to the entire sarcolemmal surface and do not produce detectable cell lysis. The phospholipid classes are asymmetrically distributed: (1) the negatively charged phospholipids, PS/PI are located exclusively in the inner or cytoplasmic leaflet; (2) 75% of PE is in the inner leaflet; (3) 93% of sphingomyelin is in the outer leaflet; (4) 43% of PC is in the outer leaflet. The predominance of PS/PI and PE at the cytoplasmic sarcolemmal surface is discussed with respect to phospholipid-ionic binding relations between phospholipids and exchange and transport of ions, and the response of the cardiac cell on ischemia-reperfusion.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

Isothermal titration calorimetric study of calcium association to lipid bilayers: influence of the vesicle preparation and composition

The association of Ca2+ ions with phospholipid bilayers was investigated using isothermal titration calorimetry. The study reveals that the binding enthalpy of these cations to bilayers formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) depends strongly on the method of preparation of the unilamellar vesicles. Extruded vesicles lead to an exothermic association, whereas sonicated ones lead to an endothermic association. In the later case, the calorimetric signal is sensitive to the length of the sonication period. It is proposed that a reorganization of the lipid bilayers under stress, obtained with sonicated small unilamellar vesicles, contributes to the calorimetric signal upon the titration with Ca2+. The analysis of the titrations indicates that, as expected, the nature of the association of Ca2+ with negatively charged phospholipid bilayers is essentially of electrostatic nature. Using a Scatchard approach, it is found that bilayers become saturated in Ca2+ approximately when the electroneutrality of the bilayer interface is reached. Moreover, the affinity constant was reduced by the increase of the ionic strength of the aqueous buffer. It was found that the intrinsic binding constant of Ca2+ to membranes containing 30 and 50 mol% of POPG was about 11 mM-1, in a MES buffer containing 10 mM NaCl, at pH 5.6.     

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.     

Lipopolysaccharides in bacterial membranes act like cholesterol in eukaryotic plasma membranes in providing protection against melittin-induced bilayer lysis

Melittin is a small, cationic peptide that, like many other antimicrobial peptides, lyses cell membranes by acting on their lipid bilayers. However, the sensitivity to antimicrobial peptides varies among cell types. We have performed direct binding and vesicle leakage experiments to determine the sensitivity to melittin of bilayers composed of various physiologically relevant lipids, in particular, key components of eukaryotic membranes (cholesterol) and bacterial outer membranes (lipopolysaccharide or LPS). Melittin binds to bilayers composed of both zwitterionic and negatively charged phospholipids, as well as to the highly charged LPS bilayers. The magnitude of the free energy of binding (deltaG degrees ) increases with increasing bilayer charge density; deltaG degrees = -7.6 kcal/mol for phosphatidylcholine (PC) bilayers and -8.9 to -11.0 kcal/mol for negatively charged bilayers containing phosphatidylserine (PS), phospholipids with covalently attached polyethylene glycol (PEG-lipids), or LPS. Comparisons of these data show that binding is not markedly affected by the steric barrier produced by the PEG in PEG-lipids or by the polysaccharide core of LPS. The addition of equimolar cholesterol to PC bilayers reduces the level of binding (deltaG degrees = -6.4 kcal/mol) and reduces the extent of melittin-induced leakage by 20-fold. LPS and 1:1 PC/cholesterol bilayers have similar high resistance to melittin-induced leakage, indicating that cholesterol in eukaryotic plasma membranes and LPS in Gram-negative bacteria provide strong protection against the lytic effects of melittin. We argue that this resistance is due at least in part to the similar tight packing of the lipid acyl chains in PC/cholesterol and LPS bilayers. The addition of bacterial phospholipids to LPS bilayers increases their sensitivity to melittin, helping to explain the higher sensitivity of deep rough bacteria compared to smooth phenotypes.     

Interaction of Hsp90 with phospholipid model membranes

Heat shock protein 90 (Hsp90) is an essential molecular chaperone with versatile functions in cell homeostatic control under both normal and stress conditions. Hsp90 has been found to be expressed on the cell surface, but the mechanism of Hsp90 association to the membrane remains obscure. In this study, the direct interaction of Hsp90 and phospholipid vesicles was characterized, and the role of Hsp90 on membrane physical state was explored. Using surface plasmon resonance (SPR), we observed a strong interaction between Hsp90 and different compositions of lipid. Hsp90 had a preference to bind with more unsaturated phospholipid species and the affinity was higher with negatively charged lipids than zwitterionic lipids. Increasing the mole fraction of cholesterol in the phospholipid led to a decrease of binding affinity to Hsp90. Circular dichroism (CD) spectroscopy of Hsp90 in PC membranes showed more α-helix structure than in aqueous buffer. The differential scanning calorimeter (DSC) and fluorescence polarization results showed Hsp90 could affect the transition temperature and fluidity of the bilayer. We postulate from these results that the association between Hsp90 and membranes may involve both electrostatic and hydrophobic force, and constitute a possible mechanism that modulates membrane lipid order during thermal fluctuations.     

Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects

The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.     

Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes. Evidence of affinity for negatively charged membranes.

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed of l-alpha-1,2-dimyristoylphosphatidylcholine, l-alpha-1,2-dimyristoylphosphatidylglycerol and l-alpha-1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing l-alpha-1,2-dimyristoylphosphatidylglycerol; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charged membranes is puzzling considering the previous identification of the protein as a phosphatidylethanolamine-binding protein, and suggests that the association of PEBP with phospholipid membranes is driven by a mechanism other than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the protein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEBP, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP terminal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicating that they could participate in the binding of PEBP to membranes. Our results strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.     

Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides

Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.     

Effects of verbascoside,a phenylpropanoid glycoside from lemon verbena,on phospholipid model membranes

Phenylpropanoid glycosides are water-soluble compounds widely distributed, most of them deriving from medicinal herbs. Among them, verbascoside or acteoside has exhibited a wide biological activity, being free radical scavenging the most representative one. Moreover, antitumor, antimicrobial, anti-inflammatory, anti-thrombotic and wound healing properties have been previously described. Herein, the interaction of verbascoside with phospholipid membranes has been studied by means of differential scanning calorimetry, fluorescence anisotropy and dynamic light scattering. Verbascoside showed stronger affinity for negatively charged membranes composed of phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This compound promoted phase separation of lipid domains in PC membranes and formed a stable lipid complex with and approximate phospholipid/verbascoside ratio of 4:1. Despite its hydrophilic character, verbascoside's caffeoyl moiety was located deep into the hydrophobic core of PC membranes and was almost inaccessible to spin probes located at different depths in PG membranes. This compound affected the ionization behavior of the PG phosphate group and most likely interacted with the vesicles surface. The presence of verbascoside decreased the particle size in PG unilamellar vesicles through the increase of the phospholipid head group area. A localization of verbascoside filling the upper region of PG bilayers close to the phospholipid/water interface is proposed. These effects on membranes may help to understand the mechanism of the biological activity of verbascoside and other similar phenylpropanoid glycosides.     

Activation of porcine pancreatic phospholipase A2 by the presence of negative charges at the lipid-water interface

The effect of surface charge on the porcine pancreatic phospholipase A2 catalyzed hydrolysis of organized substrates was examined through initial rate enzyme kinetic measurements. Two long-chain phospholipid substrates, phosphatidylglycerol (PG) and phosphatidylcholine (PC), were solubilized in seven detergents differing in polar head-group charge. The neutral or zwitterionic detergents selected were Triton X-100, Zwittergent 314, lauryl maltoside, hexadecylphosphocholine (C16PN), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The negatively and positively charged detergents used were cholate and CTAB, respectively. In general, the negatively charged phospholipid PG was hydrolyzed much more rapidly than the neutral (zwitterionic) phospholipid PC. The rate of hydrolysis of PG was rapid when solubilized in all the neutral detergents and in cholate but was essentially zero in the positively charged CTAB. Conversely, hydrolysis of PC was negligible when solubilized in neutral detergents, except C16PN, and was maximal in the negatively charged detergent, cholate. The rate of hydrolysis of PC solubilized in a neutral detergent became significant only when a negative surface charge was introduced by addition of SDS. Taken together, these kinetic measurements indicate that the surface charge on the lipid aggregates is an important factor in the rate of hydrolysis of phospholipid substrates and the highest activity is observed when the net surface charge is negative. Fluorescence and electron spin resonance (ESR) spectroscopic data provide additional support for this conclusion. The fluorescence emission spectrum of the single tryptophan of phospholipase A2 is a sensitive monitor of interfacial complex formation and shows that interaction of the protein with detergent micelles is strongly dependent on the presence of a negatively charged amphiphile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.     

Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.