Preferential association of apocytochrome c with negatively charged phospholipids in mixed model membranes

The mitochondrial precursor protein, apocytochrome c, binds to model membranes containing negatively charged phospholipids (Rietveld, A., Sijens, R., Verkleij, A.J. and Kruijff, B. (1983) EMBO J. 2, 907-913). In the present paper the effect of apocytochrome c on the lipid distribution in model membranes, consisting of neutral and acidic phospholipids, is examined. Both ESR and fluorescence energy transfer experiments show that the protein preferentially interacts with the negatively charged phospholipid in the mixed model membranes. Semi-quantitative analysis of the fluorescence energy transfer from the single tryptophan in apocytochrome c to the parinaric acid in phosphatidylserine or phosphatidylcholine in mixed bovine brain phosphatidylserine/egg phosphatidylcholine vesicles reveals and average donor-acceptor distance of 22-26 A and 26-30 A for phosphatidylserine and phosphatidylcholine, respectively. In addition, these experiments demonstrate that this preferential interaction does not induce the separation of large domains enriched in complexes of apocytochrome c with negatively charged phospholipids and domains enriched in neutral lipids.     

Apocytochrome c interaction with phospholipid membranes studied by Fourier-transform infrared spectroscopy.

Apocytochrome c, the heme-free precursor of cytochrome c, has been used extensively as a model to study molecular aspects of posttranslational translocation of proteins across membranes. In this report, we have used Fourier-transform infrared spectroscopy to gain further insight into the mechanism of apocytochrome c interaction with membrane phospholipids. Association of apocytochrome c with model membranes containing the acidic lipid dimyristoylphosphatidylglycerol (DMPG) as a single component results in a drastic perturbation of phospholipid structure, at the level of both the acyl chains and the interfacial carbonyl groups. However, in a binary mixture of DMPG with acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54), the perturbing effect of the protein on the acidic phospholipid is greatly attenuated. In such a membrane with mixed lipids, the physical properties of the DMPG and DMPC components are affected in a similar fashion, indicating that apocytochrome c does not induce any significant segregation or lateral-phase separation of acidic and zwitterionic lipids. Analysis of the apocytochrome c spectrum in the amide I region reveals that binding to phospholipids causes considerable changes in the secondary structure of the protein, the final conformation of which depends on the lipid to protein ratio. In the presence of a large excess of DMPG, apocytochrome c undergoes a transition from an essentially unordered conformation in solution to an alpha-helical structure. However, in complexes of lower lipid to protein ratios (less than or equal to approximately 40:1), infrared spectra are indicative of an extended, intermolecularly hydrogen-bonded beta-sheet structure. The latter is suggestive of an extensive aggregation of the membrane-associated protein.     

Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria.

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics. In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147-4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.     

Differential interactions of apo- and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria

Monomolecular layers of lipid extracts of microsomal, mitochondrial outer and inner membranes, and pure lipid species have been used to measure their interaction with apo- and holocytochrome c. Large differences were observed both with respect to the nature and the lipid specificity of the interaction. The initial electrostatic interaction of the hemefree precursor apocytochrome c with anionic phospholipids is followed by penetration of the protein in between the acyl chains. Apocytochrome c shows similar interactions for all anionic lipids tested. In strong contrast the holoprotein discriminates enormously between cardiolipin for which it has a high affinity and phosphatidylserine and phosphatidylinositol for which it has a much lower affinity. For these latter lipids the interaction with cytochrome c is primarily electrostatic. The cytochrome c-cardiolipin interaction shows several unique features which suggest the formation of a specific complex between the two molecules. These properties account for the preference in interaction of the apoprotein with the lipid extract of the outer mitochondrial membrane over that of the endoplasmic reticulum and the large preference of cytochrome c for the inner over that of the outer mitochondrial membrane lipid extract. Only apocytochrome c was able to induce close contacts between monolayers of the mitochondrial outer membrane lipids and vesicles of mitochondrial inner membrane lipids. Experiments with fragments of both protein and unfolding experiments with cytochrome c revealed that the differences in interaction between the two proteins are mainly due to differences in their tertiary structure and not the presence of the heme group itself. The initial unfolded structure of apocytochrome c is responsible for the high penetrative power of the protein and its ability to induce close membrane contact, whereas the folded structure of cytochrome c is responsible for the specific interaction with cardiolipin. The results are discussed in the light of the apocytochrome c import process in mitochondria and suggest that lipid-protein interactions contribute to targeting the precursor toward mitochondria and are important for its translocation across the outer mitochondrial membrane and the final localization of cytochrome c toward the outside of the inner mitochondrial membrane.     

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.     

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic and hydrophobic interactions of the intermediate filament protein vimentin and its amino terminus with lipid bilayers

Immunofluorescence and electron microscopical studies on the intracellular distribution of intermediate filaments (IFs) have demonstrated a close proximity of these cytoskeletal structures to cellular membranes. Moreover, nonepithelial IF (protein)s have been shown to exhibit high affinities for lipids, especially for negatively charged and nonpolar lipids. Here, using hydrophobic labeling with the photoactivatable phosphatidylcholine analogue [3H]1-palmitoyl-2-[11-[4-(trifluoromethyldiazirinyl]undecanoyl+ ++]-sn- glycero-3-phosphorylcholine or with 1-azidopyrene at low and physiological ionic strength, it is demonstrated that the IF subunit protein vimentin can interact with the hydrophobic core of lipid bilayers, in addition to strong ionic relationships between both reactants. Whereas the presence of acidic phospholipids in the lipid vesicles was absolutely essential for efficient vimentin labeling, cholesterol played a synergistic role in this reaction. Proteolytic degradation of photolabeled vimentin localized the derivatization exclusively to the non-alpha-helical, highly positively charged N-terminal domain of the filament protein. Furthermore, circular dichroism studies performed on the isolated N terminus of vimentin revealed a significant increase in the alpha-helical content of the polypeptide upon its interaction with vesicles containing negatively charged phospholipids. These results indicate an amphiphilic character of the N terminus and suggest that the cationic arginine residues of the N-terminal domain react with the negatively charged head groups of acidic phospholipids prior or parallel to interaction of the polypeptide with hydrophobic regions of the lipid bilayer.     

Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.     

Lipid-protein interactions in membranes

The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy. The ESR spectra reveal a reduction in mobility of the spin-labelled lipid species, and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria. Integral proteins induce a more direct motional restriction of the spin-labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiolipin associated with the mitochondrial ADP-ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM-20 mutant, and the difference in lipid-protein interactions with the beta-sheet and alpha-helical conformations of the M-13 coat protein, has been characterized.     

Is the mitochondrial precursor protein apocytochrome c able to pass a lipid barrier?

To obtain insight into the role of lipids in the translocation of extramitochondrially synthesized proteins, we studied the ability of apocytochrome c to pass lipid bilayers. With polyacrylamide gel electrophoresis, the digestion of externally added apocytochrome c by trypsin, enclosed in lipid vesicles, was followed. The experiments demonstrate that apocytochrome c is able to pass a lipid barrier and this process shows both a lipid- and protein specificity. The most probable molecular mechanisms involved in this phenomenon are discussed.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Interaction of cytochrome c and its precursor apocytochrome c with various phospholipids

The effects of cytochrome c and apocytochrome c on the structural properties of various membrane phospholipids in model systems were compared by binding, calorimetric, permeability, ³¹P n.m.r. and freeze-fracture experiments. Both cytochrome c and apocytochrome c experience strong interactions only with negatively charged phospholipids; apocytochrome c interacted more strongly than cytochrome c. These interactions are primarily electrostatic but also have a hydrophobic character. Cytochrome c as well as apocytochrome c induces changes in the structure of cardiolipin liposomes as is shown by ³¹P n.m.r. and freeze-fracture electron microscopy. Cytochrome c does not affect the bilayer structure of phosphatidylserine. In contrast, interaction of apocytochrome c with this phospholipid results in changes of the ³¹P n.m.r. bilayer spectrum of the liposomes and also particles are observed at the fracture faces. The results are discussed in relation to the import of the protein into the mitochondrion.     

Modulation of membrane surface curvature by peptide-lipid interactions

Recent reports on the interaction of cardiotoxin and melittin with phospholipid model membranes are reviewed and analyzed. These types of peptide toxins are able to modulate lipid surface curvature and polymorphism in a highly lipid-specific way. It is demonstrated that the remarkable variety of effects of melittin on the organization of different membrane phospholipids can be understood in a relatively simple model, based on the shape-structure concept of lipid polymorphism and taking into account the position of the peptide molecule with respect to the lipids. Based on the strong preference of the peptides for negatively charged lipids and the structural consequences thereof, and on preliminary studies of signal peptide-lipid interaction, a role of inverted or concave lipid structures in the process of protein translocation across membranes is suggested.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.

Apocytochrome c derived from horse heart cytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region of the primary sequence, and cytochrome c from yeast was spin-labeled on the single cysteine residue at sequence position 102 in the C-terminal region. The spin-labeled apocytochrome c and cytochrome c were bound to fluid bilayers composed of different negatively charged phospholipids that also contained phospholipid probes that were spin-labeled either in the headgroup or at different positions in the sn-2 acyl chain. The location of the spin-labeled cysteine residues on the lipid-bound proteins was determined relative to the spin-label positions in the different spin-labeled phospholipids by the influence of spin-spin interactions on the microwave saturation properties of the spin-label electron spin resonance spectra. The enhanced spin relaxation observed in the doubly labeled systems arises from Heisenberg spin exchange, which is determined by the accessibility of the spin-label group on the protein to that on the lipid. It is found that the labeled cysteine groups in horse heart apocytochrome c are located closest to the 14-C atom of the lipid acyl chain when the protein is bound to dimyristoyl- or dioleoyl-phosphatidylglycerol, and to that of the 5-C atom when the protein is bound to a dimyristoylphosphatidylglycerol/dimyristoylphosphatidylcholine (15:85 mol/mol mixture. On binding to dioleoylphosphatidylglycerol, the labeled cysteine residue in yeast cytochrome c is located closest to the phospholipid headgroups but possibly between the polar group region and the 5-C atom of the acyl chains. These data determine the extent to which the different regions of the proteins are able to penetrate negatively charged phospholipid bilayers.     

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the sn-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with phosphatidylserine dispersions containing lipids spin-labeled at the C12 or C14 position [G?rrissen, H., Marsh, D., Rietveld, A., & de Kruijff, B. (1986) Biochemistry 25, 2904-2910], was observed both on binding the carboxy-terminal fragments and on binding of the amino-terminal fragments of the precursor protein. Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy- and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.     

Interaction of the pore-forming protein equinatoxin II with model lipid membranes: A calorimetric and spectroscopic study.

The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry. EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition. This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10. For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule. The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements. Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG). The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII. The second transition involves the formation of alpha-helical structure. Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association.     

The sterol carrier protein-2 amino terminus: a membrane interaction domain.

Sterol carrier protein-2 (SCP2) is a small, 123 amino acid, protein postulated to play a role in intracellular transport and metabolism of lipids such as cholesterol, phospholipids, and branched chain fatty acids. While it is thought that interaction of SCP2 with membranes is necessary for lipid transfer, evidence for this possibility and identification of a membrane interaction domain within SCP2 has remained elusive. As shown herein with circular dichroism and a direct binding assay, SCP2 bound to small unilamellar vesicle (SUV) membranes to undergo significant alteration in secondary structure. The SCP2 amphipathic N-terminal 32 amino acids, comprised of two alpha-helical segments, were postulated to represent a putative phospholipid interaction site. This hypothesis was tested with a series of SCP2 N-terminal peptides, circular dichroism, and direct binding studies. The SCP2 N-terminal peptide (1-32)SCP2, primarily random coil in aqueous buffer, adopted alpha-helical structure upon interaction with membranes. The induction of alpha-helical structure in the peptide was maximal when the membranes contained a high mole percent of negatively charged phospholipid and of cholesterol. While deletion of the second alpha-helical segment within this peptide had no effect on formation of the first alpha-helix, it significantly weakened the peptide interaction with membranes. Substitution of Leu(20) with Glu(20) in the N-terminal peptide disrupted the alpha-helix structure and greatly weakened the peptide interaction with membranes. Finally, deletion of the first nine nonhelical amino acids had no effect either on formation of alpha-helix or on peptide binding to membranes. N-Terminal peptide (1-32)SCP2 competed with SCP2 for binding to SUV. These data were consistent with the N-terminus of SCP2 providing a membrane interaction domain that preferentially bound to membranes rich in anionic phospholipid and cholesterol.     

The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the ternary system ReLPS/DMPEd54/PLL(500) showed the features of a phase segregation phenomenon not affecting the transition temperatures of the pure lipid components, the ternary system composed of ReLPS/DMPEd54 and OmpF exhibited phase transition curves that were characterized by an unspecific (DMPEd54/OmpF) and a strong and unique (ReLPS/OmpF) type of lipid-protein interaction. Furthermore, semiquantitative estimations supported the supposition that OmpF might be able to induce bilayer asymmetry in preformed symmetrical ReLPS/DMPEd54 vesicles.     

A molecular dynamics study of the interaction between domain I-BAR of the IRSp53 protein and negatively charged membranes

The methods of computer simulation in all-atom and coarse-grained approximations have been used to study specific interactions of the isolated domain I-BAR of the actin-binding protein IRSp53 with model membranes containing neutral phospholipids and those including negatively charged PI(4,5)P₂ phospholipids. It has been shown that the I-BAR domain does not interact with neutral lipids but induces bending of the synthetic membrane rich in negatively charged phospholipids. Clustering of charged lipids on the surface of the membrane at the sites of its interaction with the protein has been observed. This indicates that the interaction of I-BAR with negatively charged lipids is of electrostatic and hydrophobic nature.