Presence Of Phosphatidylcholine Vesicles Research Articles

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8A resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp --> Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.

Promotion of peptide antimicrobial activity by fatty acid conjugation.

Three peptides, YGAA[KKAAKAA](2) (AKK), KLFKRHLKWKII (SC4), and YG[AKAKAAKA](2) (KAK), were conjugated with lauric acid and tested for the effect on their structure, antibacterial activity, and eukaryotic cell toxicity. The conjugated AKK and SC4 peptides showed increased antimicrobial activity relative to unconjugated peptides, but the conjugated KAK peptide did not. The circular dichroism spectrum of AKK showed a significantly larger increase in its alpha-helical content in the conjugated form than peptide KAK in a solution containing phosphatidylethanolamine/phosphotidylglycerol vesicles, which mimics bacterial membranes. The KAK and AKK peptides and their corresponding fatty acid conjugates showed little change in their structure in the presence of phosphatidylcholine vesicles, which mimic the cell membrane of eukaryotic cells. The hemolytic activity of the KAK and AKK peptides and conjugates was low. However, the SC4 fatty acid conjugate showed a large increase in hemolytic activity and a corresponding increase in helical content in the presence of phosphatidylcholine vesicles. These results support the model of antimicrobial peptide hemolytic and antimicrobial activity being linked to changes in secondary structure as the peptides interact with lipid membranes. Fatty acid conjugation may improve the usefulness of peptides as antimicrobial agents by enhancing their ability to form secondary structures upon interacting with the bacterial membranes.

Cross-linking Phosphatidylinositol-specific Phospholipase C Traps Two Activating Phosphatidylcholine Molecules on the Enzyme

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein. The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.

Determination of lipid bilayer/water partition coefficient of new phenothiazines using the second derivative of absorption spectra method.

The partition coefficients (K(p)) between lipid bilayer of phosphatidylcholine (PC) vesicles and buffer for five new phenothiazines were determined using the second derivatives of ultraviolet absorption spectra. The lambda(max) of absorption band for each of the investigated phenothiazine derivatives (PDs) was shifted to the longer wavelengths in the presence of PC vesicles with increasing of lipid concentration. As a result of light scattering in liposome suspension no isosbestic point could be observed in absorption spectra. However, the background signal could be eliminated using the method of second derivative of absorption spectra. In the second derivative of absorption spectra two isosbestic points were observed. Changes of intensity (Delta D) of second derivative of absorption spectra at the lambda(max) (wavelength of absorption maximum for drug in buffer) caused by the increase in lipid concentration were measured for set of phenothiazine derivatives. K(p) for these drugs were calculated from the relationship between Delta D and lipid concentration. The K(p) values for all studied phenothiazine derivatives are in the order of magnitude of 10(5) and they increase about 1.7-fold when length of the alkyl phenothiazine chain was enhanced by addition of the each next one (-CH(2)) group. Substitution of -H atom by -CF(3) group at position 2 of phenothiazine ring results in 3.5-fold increase in K(p) values.

Dynorphin-phospholipid membrane interactions: role of phospholipid head-group and cholesterol.

The interaction of the kappa-opioid receptor-selective heptadecapeptide dynorphin A(1-17) (Tyr1-Gly-Gly-Phe-Leu5-Arg-Arg-Ile-Arg-Pro10-Lys-Leu-Lys-Trp-As p15-Asn-Glu) with phospholipid membranes has been investigated by monitoring the leakage of the internal aqueous contents of liposomes, the changes in the tryptophan emission spectrum, and the collisional quenching of tryptophan fluorescence by brominated lipids. The peptide induces more extensive leakage of contents from phosphatidylserine than from phosphatidylcholine vesicles, and experiences a blue shift of the Trp fluorescence emission maximum in the presence of phosphatidylserine vesicles. In the presence of phosphatidylcholine vesicles, however, the Trp fluorescence intensity is reduced without a blue shift. In phosphatidylserine membranes containing 10 mol% phosphatidylcholine, the intensity of the blue-shifted fluorescence is enhanced. This avid interaction of dynorphin A(1-17) with phosphatidylserine membranes is likely to be mediated by the positively charged Arg and Lys groups. It is proposed that, while the N-terminus of the peptide may be embedded in the bilayer in analogy with dynorphin (1-13), the C-terminal region of dynorphin A (1-17) bends back onto the bilayer/water interphase, and that the Trp14 residue is stabilized in a hydrophobic pocked near the interphase by the interaction of the neighboring charged amino acids with the phosphate, carboxyl and amino groups on phosphatidylserine.

Fluorescence analysis of the interaction between ganglioside G M1-containing phospholipid vesicles and the B subunit of cholera toxin

Binding of cholera toxin B protomer (CT-B) to a pyrene-labeled analogue of its ganglioside G M1 receptor (pyrene-G M1) in the absence and presence of phosphatidylcholine vesicles was monitored using steady-state fluorescence spectroscopy. CT-B association with pyrene-G M1 micelles induces changes in the fluorescence properties of this ganglioside analogue that are consistent with its conversion from an excimer to a monomer form. Incubation of pyrene-G M1 with preformed vesicles of phosphatidylcholine (PC) results in complete conversion of pyrene-G M1 to its monomer form, however, unlike with CT-B binding, incorporation of pyrene-G M1 into PC vesicles occurs with a concomitant loss of fluorescence quenching by the small polar quenching agent acrylamide. Subsequent binding of CT-B to the PC-G M1 composite vesicles causes no further change in the pyrene fluorescence emission spectrum but does appear to increase acrylamide accessibility. These data lead to the conclusion that cholera toxin binding to a cell membrane alters membrane packing at the site of attachment. Furthermore, this phenomenon appears to be influenced by environmental conditions such as pH. A pH of about 4.0 or less causes acrylamide quenching to decrease to approximately the levels observed in the absence of CT-B. These results may be useful in describing the dynamics of the interaction between cholera toxin and target cell membranes. Moreover, these data could provide clues to the mechanism by which the toxic portion of CT is able to enter the cytoplasm of target cells.

Structure and orientation of a bilayer-bound model tripeptide: a proton NMR study

The conformation and orientation of a model peptide, A-F-A-O-tert-butyl, was studied in sonicated unilamellar phosphatidylcholine (PC) vesicles by proton NMR. Intramolecular nuclear Overhauser enhancements (NOEs) were measured for the tripeptide in free solution and with PC vesicles. The peptide has no stable tertiary structure in free solution but adopts a structure in the presence of PC vesicles. Restrained molecular dynamics using NOE constraints were used to deduce a preferred structure for the bound tripeptide

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent [formula omitted

The hydrophobic photolabel 3-( trifluoromethyl)-3-(m- [ 125 I] iodophenyl) diazirine([ 125 I] TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2–4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5–7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.

Formation of protein polymers in erythrocyte ghosts incubated with sonicated lipid vesicles. Effects on spectrin extractibility, permeability of ghosts to vesicles, intramembrane particle distribution and bleb formation

The incubation of human erythrocyte white ghosts with phosphatidylcholine (PC) vesicles or cholesterol/phosphatidylcholine (C/PC) vesicles under hypotonic or isotonic conditions generated membrane protein cross-links. The latter appeared in the form of a high molecular weight polymer after SDS-polyacrylamide gel electrophoresis. The polymer started to develop within a few minutes of incubation, arising largely from spectrin, and required 24 h or more for completion. It occurred regardless of cholesterol depletion undergone by the ghosts in the presence of PC vesicles. It was not reversed upon further incubation in a hypotonic, vesicle-free medium. When initial incubation was carried out under hypotonic conditions, a number of other alterations were recorded: (i) spectrin extractibility was abolished; (ii) ghosts became gradually impermeable to vesicles within a few hours, a process referred to as slow ‘resealing’ and generating an irreversible sequestration of the vesicles; (iii) intramembrane particles aggregated and blebs free of intramembrane particles pinched off inward or outward. When initial incubation was conducted under isotonic conditions, the following was observed: (i) spectrin was unextractible, as could be expected; (ii) vesicles did not enter the ghosts, a fact indicating an immediate and complete impermeabilization of ghosts to vesicles, a process referred to as fast ‘resealing’; (iii) intramembrane particle aggregation and blebs free of intramembrane particles were also present. When initial incubation was performed under isotonic conditions, but in the absence of vesicles, the polymer failed to be associated with spectrin inextractibility. These data support the view that lipid vesicles generate a high molecular weight polymer-associated, slow resealing of erythrocyte ghosts that differs, at least in part, from the polymer-free, fast resealing induced by a vesicle-free isotonic medium. Resistance to β-mercaptoethanol of the polymer makes unlikely the sole participation of disulfide bonds. Absence of added Ca 2+ in the medium is inconsistent with the transglutaminase-catalyzed formation of amide linkages. When ghosts were separated from the vesicles by a cellophane membrane upon hypotonic incubation, spectrin remained extractible and no polymer developed. Sonication of the vesicles under nitrogen and in the presence of butylated hydroxytoluene did not prevent the formation of the polymer.

Calcium-induced interaction of phospholipid vesicles and bilayer lipid membranes

The interaction of unilamellar phospholipid vesicles with bilayer lipid membranes has been studied by observing the electrical conductance of the planar membrane. The presence of phosphatidylcholine vesicles as well as phosphatidylcholine/phosphatidylserine (1 : 1) vesicles on one side compartment of the bilayer membrane, but not phosphatidylserine vesicles, causes discrete fluctuations in the phosphatidylserine membrane conductance, which is also increased by at least an order of magnitude. These events are dependent on vesicle concentration as well as the presence of Ca 2+. The results are interpreted in terms of the incorporation of domains of phosphatidylcholine into the membrane, which confer a higher conductance state.