An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

The amyloid precursor protein interacts with neutral lipids.

The amyloid protein precursor (APP) was incorporated into liposomes or phospholipid monolayers. APP insertion into liposomes required neutral lipids, such as L-alpha-phosphatidylcholine, in the target membrane. It was prevented in vesicles containing L-alpha-phosphatidylserine. The insertion was enhanced in acidic solutions, suggesting that it is modulated by specific charge/charge interactions. Surface-active properties and behaviour of APP were characterized during insertion of the protein in monomolecular films of L-alpha-phosphatidylcholine, L-alpha-phosphatidylethanolamine or L-alpha-phosphatidylserine. The presence of the lipid film enhanced the rate of adsorption of the protein at the interface, and the increase in surface pressure was consistent with APP penetrating the lipid film. The adsorption of APP on the lipid monolayers displayed a significant head group dependency, suggesting that the changes in surface pressure produced by the protein were probably affected by electrostatic interactions with the lipid layers. Our results indicate that the penetration of the protein into the lipid monolayer is also influenced by the hydrophobic interactions between APP and the lipid. CD spectra showed that a large proportion of the alpha-helical secondary structure of APP remained preserved over the pH or ionic strength ranges used. Our findings suggest that APP/membrane interactions are mediated by the lipid composition and depend on both electrostatic and hydrophobic effects, and that the variations observed are not due to major secondary structural changes in APP. These observations may be related to the partitioning of APP into membrane microdomains.     

Parameters modulating the maximum insertion pressure of proteins and peptides in lipid monolayers

The lipid monolayer model membrane is useful for studying the parameters responsible for protein and peptide membrane binding. Different approaches have been used to determine the extent of protein and peptide binding to lipid monolayers. This review focuses on the use of the “maximum insertion pressure” (MIP) to estimate the extent of protein and peptide penetration in lipid monolayers. The MIP data obtained with different proteins and peptides have been reviewed and discussed which allowed to draw conclusions on the parameters modulating the monolayer binding of proteins and peptides. In particular, secondary structure components such as amphipathic α-helices of proteins and peptides as well as electrostatic interactions play important roles in monolayer binding. The MIPs have been compared to the estimated lateral pressure of biomembranes which allowed to evaluate the possible association between proteins or peptides with natural membranes. For example, the MIP of a membrane-anchored protein with a glycosylphosphatidylinositol (GPI) was found to be far below the estimated lateral pressure of biomembranes. This allowed us to conclude that this protein is probably unable to penetrate the membrane and should thus be hanged at the membrane surface by use of its GPI lipid anchor. Moreover, the values of MIP obtained with myristoylated and non-myristoylated forms of calcineurin suggest that the myristoyl group does not contribute to monolayer binding. However, the acylation of a peptide resulted in a large increase of MIP. Finally, the physical state of lipid monolayers can have a strong effect on the values of MIP such that it is preferable to perform measurements with lipids showing a single physical state. Altogether the data show that the measurement of the maximum insertion pressure provides very useful information on the membrane binding properties of proteins and peptides although uncertainties must be provided to make sure the observed differences are significant.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study

(1) The interaction of apocytochrome c with different molecular species of phosphatidylserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 mN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidylserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidylserine is indicated by the low Kd of 0.017 microM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using 14C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interfacial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 A2/molecule in the absence of lipid to 700-900 A2/molecule after penetration of dioleoylphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidylserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.     

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.     

Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b.     

Activation of protein kinase C in lipid monolayers

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.     

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

Interaction of Doxorubicin with phospholipid monolayer and liposomes

The effect of Doxorubicin which is (an anthracycline antibiotic with a broad spectrum of antitumor activity) on the monolayer and bilayer in the form of large Multilamellar Vesicles (MLV's) of Dipalmitoyl phosphatidylcholine (DPPC) were studied by means of monolayer techniques (surface pressure, penetration kinetics, and association constant) and light scattering technique. The monolayer technique showed that addition of DXR to a lipid film composed of (DPPC/CHOL/PEG-PE) at a molar ratio of (100:0:0) produced a less condensed Monolayer. In the (π-A) curves, DXR induced shift towards larger area/molecule, where the area/molecule was shifted from 61 to 89 A², and 116 A² in the presence of 20 and 40 nM DXR, respectively. The three curves collapsed at a pressure π = 45 mN/m. In penetration kinetics experiment (Δπ-t), the change in pressure with time was 8 and 14 mN/m for a DXR concentration of 20 and 40 nM, respectively, and the increase in surface pressure presented a plateau over a period of 30 min. The measured association constant (K) was found to be 5 × 10⁵/M. In the light scattering experiment, there was a shift of the transition temperature (T_m) of (MLV's) of the same composition of the monolayer towards a smaller value from 40.5° to 34.5°C. Incorporation of CHOL and PEG-PE as DPPC/CHOL/PEG-PE at a molar ratio of (100:20:0), (100:0:4) and (100:20:4) greatly counteracted the effect of DXR and made the lipid membrane more condense and rigid. Moreover, the penetration of DXR into the membrane was greatly reduced. There was a very small shift for the (π-A) and (Δπ-t) curves, and the association constant of the drug for these different lipid compositions was greatly reduced down to 2.5 × 10⁵/M and the transition temperature (T_m) was increased up to (42.5°C) in the presence of 40 nM DXR. Our results suggest that DXR has a great effect on the phospholipid membrane, and that addition of CHOL or PEG-PE to the phospholipid membrane causes stabilization for the membrane, and reduces the interaction with Doxorubicin.     

The reactivities of tyrosine and tryptophan residues in lipid-bound cytochrome b5.

Purified cytochrome b5 from rabbit liver microsomes was bound to liposomes prepared from microsomal lipids. Tyrosyl and tryptophyl side chains of the protein were modified by water-soluble reagents and the reactivities of these amino acid residues in the liposome-bound cytochrome b5 were compared to those of the free protein. At pH 13, 80% of the tyrosines in lipid-free cytochrome b5 ionized immediately, whereas in the lipid-bound protein only 65% ionized within the first minute. In contrast, acetylation with acetylimidazole resulted in the conversion of all 5 tyrosine groups of lipid-free as well as lipid-bound cytochrome b5 into O-acetylated derivatives, which upon treatment with hydroxylamine were completely deacetylated. Reaction with N-bromosuccinimide revealed that only 60% of the 4 tryptophan residues present in cytochrome b5 were accessible to the reagent in the lipid-bound protein, although all tryptophans could be modified in lipid free cytochrome b5. It was concluded that the two tyrosines in the region linking the protein to the membrane are not shielded by lipid bilayer but that of the three tryptophans in the same region one is completely buried in the membrane, whereas the remaining two tryptophans may be both partly exposed to the solvent or alternatively, one may be partially and the other completely exposed.     

An analysis of the interaction of protein with lipid monolayers at the air/water interface

1. Measurements have been made of the interaction of cytochrome c, bovine serum albumin and synthetic oxytocin with low-pressure (2dyn/cm) monolayers of stearic acid, phosphatidylcholine and phosphatidylethanolamine. 2. [(14)C]Carboxymethylation of the cytochrome c and albumin followed by surface-radioactivity determinations have shown that only a proportion of the protein added to the subphase is bound to the monolayers and that initially the degree of binding is dependent on the protein concentration. The binding is irreversible in the sense that the adsorbed protein cannot be removed by transferring the film containing the interacted protein to a fresh subphase containing no protein. 3. Three successive types of interaction can usually be recognized. (a) Initially, whole molecules of protein penetrate the lipid film and occupy the same area as those of the protein spread at the air/water interface. (b) Above certain film pressures a part of each protein molecule, probably hydrophobic side chains, penetrates the film. The change in surface pressure per unit of bound protein is much smaller than in (a). (c) At higher film pressures, adsorption without penetration occurs. With cytochrome c this is initially dependent on a favourable electrostatic interaction.     

Effects of proteins on thermotropic phase transitions of phospholipid membranes.

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three catagories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion. Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (deltaH) accompanied by either an increase or no change in the temperature of transition (Tc). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region. Cytochrome c and A1 protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both Tc and deltaH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer. Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the Tc but did induce a linear decrease in the magnitude of the deltaH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a deltaH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.     

Molecular mechanisms of apoptosis. Structure of cytochrome c-cardiolipin complex

One of the functions of cytochrome c in living cells is the initiation of apoptosis by catalyzing lipid peroxidation in the inner mitochondrial membrane, which involves cytochrome c bound with acidic lipids, especially cardiolipin. In this paper the results of studies of cytochrome c-cardiolipin complex structure carried out by different authors mainly on unilamellar cardiolipin-containing phospholipid liposomes are critically analyzed. The principal conclusion from the published papers is that cytochrome c-cardiolipin complex is formed by attachment of a cytochrome c molecule to the membrane surface via electrostatic interactions and the subsequent penetration of one of the fatty-acid cardiolipin chains into the protein globule, this being associated with hydrophobic interactions that break the >Fe…S(Met80) coordinate bond and giving rise to appearance of cytochrome c peroxidase activity. Nevertheless, according to data obtained in our laboratory, cytochrome c and cardiolipin form spherical nanoparticles in which protein is surrounded by a monolayer of cardiolipin molecules. Under the action of cooperative forces, the protein in the globule expands greatly in volume, its conformation is modified, and the protein becomes a peroxidase. In extended membranes, such as giant monolayer liposomes, and very likely in biological membranes, the formation of nanospheres of cytochrome c-cardiolipin complex causes fusion of membrane sections and dramatic chaotization of the whole membrane structure. The subsequent disintegration of the outer mitochondrial membrane is accompanied by cytochrome c release from the mitochondria and triggering of a cascade of programmed cell death reactions.     

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)     

A thermodynamic and structural study of myelin basic protein in lipid membrane models

Myelin basic protein (MBP) is a major protein of the myelin membrane in the central nervous system. It is believed to play a relevant role in the structure and function of the myelin sheath and is a candidate autoantigen in demyelinating processes such as multiple sclerosis. MBP has many features typical of soluble proteins but is capable of strongly interacting with lipids, probably via a conformation change. Its structure in the lipid membrane as well as the details of its interaction with the lipid membrane are still to be resolved. In this article we study the interaction of MBP with Langmuir films of anionic and neutral phospholipids, used as experimental models of the lipid membrane. By analyzing the equilibrium surface pressure/area isotherms of these films, we measured the protein partition coefficient between the aqueous solution and the lipid membrane, the mixing ratio between protein and lipid, and the area of the protein molecules inserted in the lipid film. The penetration depth of MBP in the lipid monolayer was evaluated by x-ray reflectivity measurements. The mixing ratio and the MBP molecular area decrease as the surface pressure increases, and at high surface pressure the protein is preferentially located at the lipid/water interface for both anionic and neutral lipids. The morphology of MBP adsorbed on lipid films was studied by atomic force microscopy. MBP forms bean-like structures and induces a lateral compaction of the lipid surface. Scattered MBP particles have also been observed. These particles, which are 2.35-nm high, 4.7-nm wide, and 13.3-nm long, could be formed by protein-lipid complexes. On the basis of their size, they could also be either single MBP molecules or pairs of c-shaped interpenetrating molecules.