Influence of lipid chain unsaturation on melittin-induced micellization.

It is well known that melittin, an amphipathic helical peptide, causes the micellization of phosphatidylcholine vesicles. In the present work, we conclude that the extent of micellization is dependent on the level of unsaturation of the lipid acyl chains. We report the results obtained on two systems: dipalmitoylphosphatidylcholine (DPPC), containing 10(mol)% saturated or unsaturated fatty acid (palmitic, oleic, or linoleic), and DPPC, containing 10(mol)% positively charged diacyloxy-3-(trimethylammonio)propane bearing palmitic or oleic acyl chains. For both systems, the presence of unsaturation in the lipid acyl chains inhibits melittin-induced micellization. Conversely, the addition of saturated palmitic acid to the DPPC matrix enhances the micellization. This modulation is proposed to be associated with the cohesion of the hydrophobic core. When the lipid chain packing of the gel-phase bilayer is already perturbed by the presence of unsaturation, it seems easier for the membrane to accommodate melittin at the interface, and the distribution of the peptide in the bilayer could be the origin of the inhibition of the micellization. The cohesion of the apolar core is shown to play an unquestionable role in melittin-induced micellization; however, this contribution does not appear to be as important as the electrostatic interactions between melittin and positively or negatively charged lipids.     

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.     

Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.     

Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization

In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.     

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (k(a)) and dissociation (k(d)) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-alpha-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high alpha-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones.     

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.     

Investigation of the interaction between melittin and dipalmitoylphosphatidylglycerol bilayers by vibrational spectroscopy.

Melittin is shown to affect the structure of the charged phospholipid dipalmitoylphosphatidylglycerol (DPPG). In the gel phase, the presence of melittin leads to (i) an increased lipid interchain vibrational coupling, (ii) a shift of the rectangular to hexagonal lipid packing transition toward low temperatures, (iii) a very small conformational disordering effect, (iv) a decrease of the polarity or hydrogen bonding capability of the lipid ester group surrounding, (v) an important decrease of the water content in the complexes where the remaining water has a more disordered structure than bulk water, and (vi) an interlamellar repeat distance of 79 A. All these observations are rationalized by the following model: adjacent bilayers of DPPG are bridged by tetramers of melittin through electrostatic interactions inducing surface charge neutralization and partial dehydration of the complexes. Melittin also affects the thermotropic behavior of DPPG. When a small amount of the toxin is present, its affinity for charged lipids is such that a phase separation occurs, the domains being stable enough to have their own gel to liquid-crystalline phase transition. In the fluid state, a deeper penetration into the lipid matrix is proposed based on the downshift of the phase transition and the low vibrational interchain coupling. This study brings out general features of cationic species/anionic lipid complexes. The charge neutralization leads to stronger interchain coupling, and electrostatic bridging of adjacent bilayers seems to be common. The hydrophobicity of the peptide is a key factor in the modulation of the gel to liquid-crystalline phase transition and in its insertion in the fluid lipid matrix.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Osmotic and pH transmembrane gradients control the lytic power of melittin.

Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes.     

Penetratin-membrane association: W48/R52/W56 shield the peptide from the aqueous phase

Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a pi-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate.     

Effect of variations in the structure of a polyleucine-based alpha-helical transmembrane peptide on its interaction with phosphatidylglycerol bilayers

High-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy were used to study the interaction of a cationic alpha-helical transmembrane peptide, acetyl-Lys(2)-Leu(24)-Lys(2)-amide (L(24)), and members of the homologous series of anionic n-saturated diacyl phosphatidylglycerols (PGs). Analogues of L(24), in which the lysine residues were replaced by 2,3-diaminopropionic acid (L(24)DAP), or in which a leucine residue at each end of the polyleucine sequence was replaced by a tryptophan (WL(22)W), were also studied to investigate the roles of lysine side-chain snorkeling and aromatic side-chain interactions with the interfacial region of phospholipid bilayers. The gel/liquid-crystalline phase transition temperature of the host PG bilayers is altered by these peptides in a hydrophobic mismatch-dependent manner, as previously found with zwitterionic phosphatidylcholine (PC) bilayers. However, all three peptides reduce the phase transition temperature and enthalpy to a greater extent in anionic PG bilayers than in zwitterionic PC bilayers, with WL(22)W having the largest effect. All three peptides form very stable alpha-helices in PG bilayers, but small conformational changes are induced in response to a mismatch between peptide hydrophobic length and gel-state lipid bilayer hydrophobic thickness. Moreover, electrostatic and hydrogen-bonding interactions occur between the terminal lysine residues of L(24) and L(24)DAP and the polar headgroups of PG bilayers. However, such interactions were not observed in PG/WL(22)W bilayers, suggesting that the cation-pi interactions between the tryptophan and lysine residues predominate. These results indicate that the lipid-peptide interactions are affected not only by the hydrophobic mismatch between these peptides and the host lipid bilayer, but also by the tryptophan-modulated electrostatic and hydrogen-bonding interactions between the positively charged lysine residues at the termini of these peptides and the negatively charged polar headgroups of the PG bilayers.     

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

Lipids Modulate the Increase of BK Channel Calcium Sensitivity by the β1 Subunit

Co-expression of the auxiliary β1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of β1′s interaction with α. We take a novel approach, exploring whether β1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+β1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14∶1), C (18∶1), C (22∶1) and C (24∶1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the β1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca²⁺ sensitivity by β1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of β1 reduces BK activity in thin bilayers of PC 14∶1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18∶1, PC 22∶1 and PC 24∶1 bilayers. These data suggest that auxiliary β1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions.     

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger.

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 +/- 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was approximately 10(6) s(-1), at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.