Lipid-protein interactions in membrane models fluorescence polarization study of cytochrome b5-phospholipids complexes

According to previous authors, cytochrome $\text{b}{}_5$, when extracted from bovine liver by a detergent method, is called cytochrome $\text{d-b}{}_5$. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome $\text{t-b}{}_5$.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, $\text{R}$, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome $\text{d-b}{}_5$, whereas $\text{R}$ does not change for cytochrome $\text{c}$ and cytochrome $\text{t-b}{}_5$. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome $\text{d-b}{}_5$ occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome $\text{d-b}{}_5$, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

α-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid.The enthalpy changes associated with the interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (ΔH) are not only larger than at physiological pH, but also show a maximum at about 23°C in the ΔH vs. temperature graph. At pH 6 and 7, on the contrary, ΔH increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of α-lactalbumin, while at pH 4 and 5 at 23°C an extra peak appears in the gel-filtration graphs between the pure vesicles and α-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23°C and a blue shift at 37°C, both to 336 nm, are observed for λ_max of the fluorescence emission spectra at pH 4 when α-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure α-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs.The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.     

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.     

Peptide-membrane interactions A fluorescence study of the binding of oligopeptides containing aromatic and basic residues to phospholipid vesicles

The binding of oligopeptides containing basic and aromatic residues to phospholipid vesicles has been studied by fluorescence spectroscopy. Tryptophan-containing peptide such as Lys-Trp-Lys or Lys-Trp(OMe) exhibit a shift of their fluorescence toward shorter wavelengths and an increased fluorescence quantum yield upon binding to phosphatidylinositol (PI) or phosphatidylserine (PS) vesicles. No binding was detected with phosphatidylcholine vesicles. The binding is strongly dependent on ionic strength and pH. Binding decreases when ionic strength increases indicating an important role of electrostatic interactions. The pH-dependence of binding reveals that the apparent $\text{p}\text{K}$ of the terminal carboxyl group of Lys-Trp-Lys is raised by ～3 units upon binding to PI and PS vesicles. The binding of tyrosine-containing peptides to PI and PS vesicles is characterized by an increase in the fluorescence quantum yield of the peptide without any shift in fluorescence maximum. A natural nonapeptide from the myelin basic protein which contains one tryptophan residue binds to PI and PS vesicles at low pH when the acidic groups are neutralized. This binding is accompanied by a shift of the tryptophyl fluorescence toward shorter wavelengths together with an enhancement of the fluorescence quantum yield. Dissociation of the complex is achieved at high ionic strength. These results indicate that aromatic residues of oligopeptides bound to the phospholipid polar heads by electrostatic interactions become buried in a more hydrophobic environment in the vicinity of the aliphatic chains of the lipids.     

Mode of binding of cytochrome b5 to phospholipid bilayers in lamellar structure

X-ray diffraction studies were made on the multilamellar systems produced by incubation of phospholipid bilayers and the membrane protein, cytochrome b₅, or non-membrane proteins (albumin, ovalbumin and β-lactoglobulin A) at pH 8.1 in aqueous 5 mM CaCl₂ solutions.Detergent-extracted cytochrome b₅ (soluble aggregate) forms two types of lamellar phase with dipalmitoyl phosphatidylcholine bilayers, depending upon the incubation temperature. One type, which has a repeat distance of 114Å, is formed above 34°C, where the binding of cytochrome b₅ to the bilayers is hydrophobic. The other type, with a repeat distance of 153 Å, is formed below 34°C, where the binding is electrostatic. It is also suggested that cytochrome b₅ is monomeric in the former phase but remains aggregated in the latter phase.When dimyristoyl phosphatidylcholine is used, the boundary temperature for the two types shifts to 12°C. These boundary temperatures coincide with the thermal pretransition points of hydrated dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, respectively.Trypsin-treated cytochrome b₅ (monomeric) and the three non-membrane proteins exhibit only binding of the electrostatic type to the bilayers, independently of the incubation temperature. The observed repeat distances suggest that in these cases two layers of protein molecules are incorporated between the bilayers.     

Fusion of phospholipid vesicles mediated by cytochrome c

Fusion of phosphatidylserine/phosphatidylethanolamine (1/1) vesicles induced by cytochrome c is studied at a wide range of pH values. A pH profile for the fusion with maximum values at pH 5 and pH 8 is obtained and this is found to be similar to the profile for cytochrome c binding to the vesicles. The binding property of apocytochrome c to the same phospholipid vesicles is found to be about the same as that of the cytochrome c at low ionic strength, but very different at high salt concentrations. No appreciable fusion of vesicles by apocytochrome c is observed. Proteolytic treatment and dansyl chloride labeling of cytochrome c- and apocytochrome c-vesicle complexes show that the C-terminal segments of these proteins with molecular weights of about 3000 and 5000, respectively, penetrate the bilayer. The hydrophobic labeling studies with photoreactive phosphatidylcholine in the bilayer show that segments of both cytochrome c and apocytochrome c go deep into the bilayer.     

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.     

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.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

Effect of different drugs on a cytochrome c--phospholipid bilayer membrane.

Liposomes were prepared from dipalmitoyl phosphatidylcholine and dicetylphosphate and their interaction with the extrinsic membrane protein cytochrome c examined in terms of changes in 22Na permeability, electrophoretic mobility, protein binding, and motion of an incorporated spin label. The amount of cytochrome c bound displays no significant temperature dependence over the temperature range studied (from 30 to 55 degrees C) whereas cytochrome c causes an increase in 22Na efflux only above the phospholipid phase transition temperature. Interaction of the protein with the lipid vesicles causes no significant disturbance in the bilayer interior as monitored by the motion of the incorporated spin probe. The drugs 2,4-dinitrophenol and ethacrynic acid, both of which increase the magnitude of the vesicle negative charge, enhance both cytochrome c binding and its effect on 22Na permeability. In contrast, the local anesthetic dibucaine, which induces a positive surface charge on these liposomes, reduces both protein binding and the protein-induced increase in 22Na efflux. Finally, the chemicals butylated hydroxytoluene, 2-tert-butylphenol, and tert-butylbenzene, all of which cause early 'melting' of the phospholipid fatty acyl chains, block the capacity of cytochrome c to enhance 22Na permeability while having no effect on its binding to the vesicles.     

A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.     

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.

Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.     

Conformations of yeast alpha-mating factor and analog peptides as bound to phospholipid bilayer. Correlation of membrane-bound conformation with physiological activity

The transferred nuclear Overhauser effects of yeast alpha-mating factor [(1-13)peptide] in the presence of various spin-labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin-labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of alpha-mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des-Trp1-alpha-mating-factor [(2-13)peptide] in the membrane-bound state has been found to be similar to that of (1-13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1-13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main-chain conformations of these three analogs in the membrane-bound state have been found to be similar to that of (1-13)peptide, although the side-chain conformations of the D-amino acid residues are naturally different from those of the L-amino acid ones. Thus, the physiological activities of the (1-13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane-bound state.     

Influence of proteins on the reorganization of phospholipid bilayers into large domains.

Using large (5-10 microns) vesicles formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera, and digital image processor, we examined the effects of membrane proteins on phospholipid domain formation. In vesicles composed of phosphatidic acid and phosphatidylcholine, incubation with cytochrome c induced the reorganization of phospholipids into large phosphatidic acid-enriched domains with the exclusion of phosphatidylcholine. Cytochrome c binding was demonstrated to be highest in the phosphatidic acid-enriched domain of the vesicle using the absorbance of the heme moiety for visualization. Both binding of cytochrome c and phospholipid reorganization were blocked by pretreatment of the vesicles with 0.1 M NaCl. The pore forming peptide gramicidin was examined for the effects of an integral protein on domain formation. Initially, gramicidin distributed randomly within the vesicle and showed no phospholipid specificity. Phosphatidic acid domain formation in the presence of 2.0 mM CaCl2 or 100 microM cytochrome c was not affected by the presence of 5 mol % gramicidin within the vesicles. In both cases, gramicidin was preferentially excluded from the phosphatidic acid-enriched domain and became associated with phosphatidylcholine-enriched areas of the vesicle. Thus, cytochrome c caused a major reorganization of both the phospholipids and the proteins in the bilayer.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein: a possible role in myelin formation.

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, L-alpha-dimyristoyl phosphatidylcholine and DL-alpha-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15-18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associated species. A similar interaction was observed with diacyl phosphatidylserine vesicles. These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myelin, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.

The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.     

Oxidation of cytochromes c and c2 by bacterial photosynthetic reaction centers in phospholipid vesicles. 1. Studies with neutral membranes

The oxidation of cytochrome c2 by photosynthetic reaction center isolated from Rhodopseudomonas sphaeroides and incorporated into unilamellar phosphatidylcholine vesicles was found to be kinetically similar to that observed earlier for reaction centers in low detergent solution [Overfield, R.E., Wraight, C.A., & DeVault, D. (1979) FEBS Lett. 105, 137-142]. At low ionic strength the kinetics were biphasic. The fast phase indicated the formation of a cytochrome-reaction center complex with an apparent binding constant, KB, of about 10(5) M-1. However, KB decreased dramatically with increasing salt concentration, and no fast oxidation was detectable in 0.1 M NaCl. The slow cytochrome oxidation was first order in both cytochrome and reaction centers and, thus, second order overall. Deviations from theoretical second-order behavior were observed when the rate of the first-order back reaction of the primary photoproducts was significant compared to the cytochrome oxidation. This can cause serious overestimation of the second-order rate constant. The slow oxidation of cytochrome c2 by reaction centers in phosphatidylcholine vesicles exhibited a 40% lower encounter frequency than with the solubilized reaction center. This was attributed to the much lower diffusion coefficient of the reaction center in the vesicle membrane than in solution. No effects of diminished dimensionality were detected with neutral vesicles. An activation energy of 8.0 +/- 0.4 kcal x mol-1 was determined for the slow phase of cytochrome c2 oxidation by reaction centers in solution and in vesicles of several different phosphatidylcholines, including dimyristoylphosphatidylcholine above and below its phase transition temperature. Thus, the physical state of the lipid did not appear to affect any rate-limiting steps leading to cytochrome oxidation. The ionic strength dependence of the slow kinetics of oxidation of cytochromes c and c2 confirmed the electrostatic nature of the cytochrome-reaction center interaction, and the pH dependence indicated the titration of a group or groups, important to this interaction, at pH 9.5.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein. A possible role in myelin formation

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, l-α-dimyristoyl phosphatidylcholine and dl-α-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15–18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associating species.A similar interaction was observed with diacyl phosphatidylserine vesicles.These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myeling, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.