Membrane interactions of the sodium channel S4 segment and its fluorescently-labeled analogues.

A 24-amino acid peptide corresponding to the S4 segment of the sodium channel was synthesized. In order to perform fluorescence energy transfer measurements and to monitor the interaction of the peptide with lipid vesicles, the peptide was selectively labeled with fluorescence probes at either its N- or C-terminal amino acids. The fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazol-4- yl-(NBD-)labeled analogues displayed blue shifts upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. The results revealed that both the N- and C-terminus of the S4 segment are located within the lipid bilayer. Titration of solutions containing NBD-labeled peptides with SUV was used to generate binding isotherms, from which surface partition constants, in the range of 10(4) M-1, were derived. The shape of the binding isotherms as well as fluorescence energy transfer measurements suggest that aggregation of peptide monomers within the membrane readily occurs in acidic but not in zwitterionic vesicles. Furthermore, the results provide good correlation between the incidence of aggregation in PC/PS vesicles and the ability of the peptides to permeate the vesicle's membrane. However, a transmembrane diffusion potential had no detectable effect on the location of the peptide within the lipid bilayer or on its aggregation state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of D-amino acid incorporated analogues of pardaxin with membranes.

The influence of specific L- to D-amino acid substitutions on the interaction of pardaxin, a shark repellent neurotoxin polypeptide, with phospholipid vesicles and human erythrocytes is described. Twelve modified, truncated, or fluorescently labeled [with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) at their N-terminal amino acid] analogues of pardaxin were synthesized by a solid-phase method. Fluorescence measurements were used to monitor the interaction of the analogues with membranes [Rapaport, D., & Shai, Y. (1991) J. Biol. Chem. 266, 23769-23775]. Upon titration of solutions containing the NBD-labeled peptides with small unilamellar vesicles, the fluorescent emission spectra of all NBD-labeled peptides displayed similar blue-shifts, in addition to enhanced intensities, upon relocation of the probe to the more apolar environment. Binding isotherms were constructed from which surface partition constants, in the range of 10(4) M-1, were derived. The existence of an aggregation process, suggested by the shape of the binding isotherms, could be associated only with those analogues in which the N-helix (residues 1-9) was not perturbed. The alpha-helical content of the analogues was estimated by circular dichroism (CD) spectroscopy, both before and after binding to vesicles at neutral pH. The ability of the peptides to dissipate a diffusion potential and to cause calcein release, as well as to lyse human erythrocytes, served to functionally characterize the peptides. The results support a two alpha-helix model, with a bend at position 13, as best describing pardaxin in its membrane-bound state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acidic phospholipids strikingly potentiate sterol carrier protein 2 mediated intermembrane sterol transfer

A liposomal membrane model system was developed to examine the mechanism of spontaneous and protein-mediated intermembrane cholesterol transfer. Rat liver sterol carrier protein 2 (SCP2) and fatty acid binding protein (FABP, also called sterol carrier protein) both bind sterol. However, only SCP2 mediates sterol transfer. The exchange of sterol between small unilamellar vesicles (SUV) containing 35 mol % sterol was monitored with a recently developed assay [Nemecz, G., Fontaine, R. N., & Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511-541], modified to continuous polarization measurement and not requiring separation of donor and acceptor membrane vesicles. As compared to spontaneous sterol exchange, 1.5 microM rat liver SCP2 enhanced the initial rate of sterol exchange between neutral zwwitterionic phosphatidylcholine SUV 2.3-fold. More important, the presence of acidic phospholipids (2.5-30 mol %) stimulated the SCP2-mediated increase in sterol transfer approximately 35-42-fold. Thus, acidic phospholipids strikingly potentiate the effect of SCP2 by 15-18 times as compared to SUV without negatively charged lipids. Rat liver FABP (up to 60 microM) was without effect on sterol transfer in either neutral zwitterionic or anionic phospholipid containing SUV. The potentiation of SCP2 action by acidic phospholipids was suppressed by high ionic strength, neomycin, and low pH. The results suggest that electrostatic interaction between SCP2 and negatively charged membranes may play an important role in the mechanism whereby SCP2 enhances intermembrane cholesterol transfer.     

Isothermal titration calorimetry studies of the binding of a rationally designed analogue of the antimicrobial peptide gramicidin s to phospholipid bilayer membranes

The binding of the positively charged antimicrobial peptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptide-induced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a one-site binding model. The measured endothermic binding enthalpy (DeltaH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (DeltaG) of binding remains relatively constant (-8.5 to -11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TDeltaS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.     

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.     

A designed probe for acidic phospholipids reveals the unique enriched anionic character of the cytosolic face of the mammalian plasma membrane

It is generally accepted that the cytosolic face of the plasma membrane of mammalian cells is enriched in acidic phospholipids due to an asymmetric distribution of neutral and anionic phospholipids in the two bilayer leaflets. However, the phospholipid asymmetry across intracellular membranes is not known. Two models have been proposed for the selective targeting of K-Ras4B, which contains a C-terminal farnesyl cysteine methyl ester adjacent to a polybasic peptide segment, to the cytosolic face of the plasma membrane. One involves electrostatic interaction of the lipidated polybasic domain with anionic phospholipids in the plasma membrane, and the other involves binding of K-Ras4B to a specific protein receptor. To address this issue, we prepared by semi-synthesis a green fluorescent protein variant that is linked to a farnesylated, polybasic peptide corresponding to the K-Ras4B C terminus as well as a variant that contains an all-d amino acid version of the K-Ras4B peptide. As expected based on electrostatics, both constructs showed preferential in vitro binding to anionic phospholipid vesicles versus those composed only of zwitterionic phospholipid. Both constructs fully targeted to the plasma membrane when microinjected into live Chinese hamster ovary and Madin-Darby canine kidney cells. Because the all-d amino acid peptide should be devoid of binding affinity to a putative highly specific K-Ras membrane receptor, these results support an electrostatic basis for the targeting of K-Ras4B to the plasma membrane, and they support an intracellular landscape of phospholipids in which the cytosolic face of the plasma membrane is the most enriched in acidic phospholipids.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.     

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.     

Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process.     

Effective charge of melittin upon interaction with POPC vesicles

The binding of bee venom melittin to small unilamellar vesicles and large nonsonicated multilamellar bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied by means of circular dichroism, 31P-NMR and electrophoretic mobility. The melittin binding isotherm for small unilamellar vesicles (SUV) could be described by a partition equilibrium with Kp = (6 +/- 1).10(4) M-1. Electrostatic effects were taken into account by means of the Gouy-Chapman theory. Combining the partition equilibrium with the Gouy-Chapman analysis suggested an effective charge for melittin of Zp = 1.9, which is lower than the true electric charge of 5-6. The variation of the 31P-NMR signal of SUV showed the change in potential at the phosphodiester moiety of the lipid upon addition of melittin. This potential change was lower than that for an ion with an electrical charge of 5-6 and corresponded to a charge of 1.5. Electrophoretic mobility measurements with multilamellar vesicles confirmed the charge reduction effect. These experimental results show that the use of the simple Gouy-Chapman theory requires an effective electrical charge of the melittin which is lower than the formal charge.     

Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C

The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.     

Membrane insertion and lateral diffusion of fluorescence-labelled cytochrome c oxidase subunit IV signal peptide in charged and uncharged phospholipid bilayers.

The synthetic 25-residue signal peptide of cytochrome c oxidase subunit IV was labelled with the fluorophor 7-nitrobenz-2-oxa-1,3-diazole (NBD) at its single cysteine residue. Addition of small unilamellar vesicles of 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) to the labelled peptide resulted in a shift of the NBD excitation and emission spectra to shorter wavelengths. Binding of the peptide to the vesicles was measured by the increase in the fluorescence emission yield. A surface partition constant of (3.9 +/- 0.5) x 10(3) M-1 was derived from these titrations. When the membrane contained, in addition to POPC, negatively charged 1-palmitoyl 2-oleoyl phosphatidylglycerol (POPG), the NBD fluorescence spectra were further shifted to shorter wavelengths and exhibited increased quantum yields. The apparent partition constants were increased to 10(4)-10(5) M-1 for vesicles with 20 or 100 mol% POPG. Lateral diffusion of the peptide was measured by fluorescence recovery after photobleaching in multibilayers of POPC, POPG, POPC/POPG (4:1) and 1,2-dimyristoyl phosphatidylcholine. The lateral diffusion coefficients of the peptide in bilayers of POPC (8 x 10(-8) cm2/s at 21 degrees C) were 1.5-1.6-fold greater than those of NBD-labelled phospholipids (5 x 10(-8) cm2/s at 21 degrees C), but 1.5-1.8-fold smaller (3 x 10(-8) cm2/s in 20% POPG and at 21 degrees C) than the lipid diffusion coefficients in the negatively charged bilayers. It is concluded that the signal peptide associates with phospholipid bilayers in two different forms, which depend on the lipid charge. The experiments with POPC bilayers are well explained by a model in which the peptide partitions into the region of the phospholipid head-groups and diffuses along the membrane/water interface. If POPG is present in the membrane, electrostatic attractions between the basic residues of the peptide and the acidic lipid head-groups result in a deeper penetration of the bilayer. For this case, two models that are both consistent with the experimental data are discussed, in which the peptide either forms an oligomer of three to six partially helical membrane-spanning monomers, or inserts into the bilayer with its amphiphilic helical segment aligned parallel to the plane of the membrane and located near the head-group and outer hydrocarbon region of the bilayer.     

Phosphorylation reverses the membrane association of peptides that correspond to the basic domains of MARCKS and neuromodulin.

Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.     

Sidedness and chemical and kinetic properties of the vesamicol receptor of cholinergic synaptic vesicles

Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which when occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of [3H]vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N'-dicyclo-hexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles is resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. [3H]Vesamicol binding depends on deprotonation of a group of pKa1 = 6.26 +/- 0.03 and protonation of a group of pKa2 = 10.60 +/- 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analogue dl-trans-4-oxo-4-[5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl )-1- naphthalenyl]amino]butanoic acid (TPNB) is an effective inhibitor of AcCh active transport with an IC50 value of (51 +/- 8) x 10(-9) M. At 23 degrees C, [3H]vesamicol bound to the receptor at a rate of (1.74 +/- 0.06) x 10(5) M-1 s-1, and excess unlabeled vesamicol displaced a low concentration of bound [3H]vesamicol at 0.29 +/- 0.01 min-1. At 0 degrees C, 10 microM unlabeled vesamicol displaced 36 +/- 2% of a low concentration of bound [3H]vesamicol at 0.16 +/- 0.02 min-1 and 64 +/- 2% at 0.013 +/- 0.001 min-1. One micromolar unlabeled vesamicol behaved similarly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of sphingomyelins and phosphatidylcholines with fluorescent dehydroergosterol

The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.     

Binding of Crotoxin, a Presynaptic Phospholipase A2Neurotoxin, to Negatively Charged Phospholipid Vesicles

Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of less than 1 microM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to less than 1 microM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.     

Binding of a neuropeptide, substance P, to neutral and negatively charged lipids

The binding of substance P (SP), a positively charged neurotransmitter peptide, to neutral and to negatively charged phospholipids has been investigated by means of a monolayer technique. Monolayers formed at room temperature from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or mixtures of the two, were maintained throughout the course of a binding experiment at a constant surface pressure while the monolayer surface area was monitored. Injection of SP into the aqueous subphase (154 mM NaCl, 10 mM Tris adjusted to pH 7.4) led to an expansion of the monolayer surface area that was attributed to a spontaneous insertion of SP between the lipid molecules. A quantitative evaluation of the area increase at constant pressure yielded SP insertion isotherms that showed that levels of SP insertion increased directly with the monolayer POPG content and decreased to negligible levels at surface pressures above 35 +/- 1 mN/m. If electrostatic effects were ignored, these data showed biphasic behavior in Scatchard plots. The apparent binding constants ranged, at 20 mN/m, from (3.2 +/- 0.3) X 10(4) M-1 for 100% POPG monolayers to (2.0 +/- 0.05) X 10(3) M-1 for 25% POPG/75% POPC monolayers. At 32 mN/m, a monolayer surface pressure that mimics bilayer conditions, the apparent binding constant for a 100% POPG monolayer was measured to be (1.1 +/- 0.05) X 10(3) M-1. However, for a monolayer containing only 25% charged lipids, corresponding to a natural membrane composition, K app at 32 mN/m was estimated to be at most 41 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.