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1.
Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.
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X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers. 相似文献
2.
Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films. 总被引:4,自引:4,他引:0
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J M Pachence S Amador G Maniara J Vanderkooi P L Dutton J K Blasie 《Biophysical journal》1990,58(2):379-389
We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface. 相似文献
3.
The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction. 总被引:4,自引:4,他引:0
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X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films. 相似文献
4.
Incorporation of a synthetic mitochondrial signal peptide into charged and uncharged phospholipid monolayers 总被引:7,自引:0,他引:7
L K Tamm 《Biochemistry》1986,25(23):7470-7476
The interaction of the chemically synthesized 25-residue signal peptide of subunit IV of yeast cytochrome c oxidase with synthetic and natural phospholipids was studied by using a monolayer technique. Incorporation of the peptide into phospholipid monolayers was measured as surface area increase at constant surface pressure. The peptide was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into phospholipid monolayers up to limiting pressures of 30-40 mN/m. The incorporation of the positively charged peptide was strongly enhanced by the presence of negatively charged phospholipids. The molecular area of the signal peptide in monolayers was determined with a 14C-labeled signal peptide and was 560 +/- 170 A2. This is consistent with a 25-residue alpha-helical peptide incorporating with its long axis parallel to the plane of the monolayer. Incorporation isotherms into synthetic phosphatidylcholine and phosphatidylglycerol monolayers at different charge densities were analyzed in terms of a simple incorporation/binding model, involving partitioning of the peptide into the monolayer and an in-plane binding reaction of the negatively charged phospholipids to the partitioned peptide. 相似文献
5.
Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.
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Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase. 相似文献
6.
The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface. 相似文献
7.
Monolayers of charged and neutral phospholipids at the air/water interface containing the cytochromes b5 and c are studied by film balance techniques and by fluorescence microscopy. A new technique is introduced to obtain a defined and homogeneous protein distribution within the membrane. It is shown that both proteins preferentially partition into the fluid membrane phases coexisting with solid lipid domains, thus allowing formation of periodic protein distributions. Protein reconstitution in protein/lipid ratios up to 1:50 does not change the pressure, pi c, corresponding to the main lipid transition but changes the slope in the pressure/area isotherms. It also affects the pressure-induced lipid crystallization, in that the monolayer can be viewed as segregated into a protein-free and a protein-enriched phase. Whereas penetration of cytochrome c into the monolayer is highly dependent on lipid head group charge, this does not hold for cytochrome b. In both cases, monolayer penetration is monotonously reduced with increasing surface pressure, pointing to the dependence of hydrophobic protein-lipid interactions on hydrocarbon chain density. 相似文献
8.
Modulation of cytochrome C coupling to anionic lipid monolayers by a change of the phase state: a combined neutron and infrared reflection study
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The effect of monolayer domain formation on the electrostatic coupling of cytochrome c from the subphase to a monolayer at the air/water interface was studied using a combination of neutron reflection (NR) and infrared reflection absorption spectroscopy (IRRAS) techniques. The monolayers consisted of a binary mixture of the zwitterionic phosphatidylcholine and the anionic phosphatidylglycerol. For a monolayer of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG, 30 mol%), which exhibits a non-ideal mixing of the two lipid components, we observed a significantly higher protein coupling to the liquid-condensed phase compared to the liquid-expanded state. In contrast, this higher protein binding was not observed when the two lipids had identical chain lengths (nearly ideal mixing). Similarly, for an equimolar mixture of DPPC and DMPG, we did not observe significant differences in the protein binding for the two phase states. The results strongly suggest that the domain formation in a condensed monolayer under non-ideal lipid mixing conditions is crucial for the cytochrome c binding strength. Furthermore, this study demonstrates the significant advantages of gathering information on protein-monolayer coupling by the combined use of a dedicated IRRAS set-up with the NR technique. 相似文献
9.
Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy. 总被引:1,自引:0,他引:1
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The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation. 相似文献
10.
Interaction of horse heart and thermus thermophilus type c cytochromes with phospholipid vesicles and hydrophobic surfaces
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Bernad S Oellerich S Soulimane T Noinville S Baron MH Paternostre M Lecomte S 《Biophysical journal》2004,86(6):3863-3872
The binding of horse heart cytochrome c (cyt-c) and Thermus thermophilus cytochrome c(552) (cyt-c(552)) to dioleoyl phosphatidylglycerol (DOPG) vesicles was investigated using Fourier transform infrared (FTIR) spectroscopy and turbidity measurements. FTIR spectra revealed that the tertiary structures of both cytochromes became more open when bound to DOPG vesicles, but this was more pronounced for cyt-c. Their secondary structures were unchanged. Turbidity measurements showed important differences in their behavior bound to the negatively charged DOPG vesicles. Both cytochromes caused the liposomes to aggregate and flocculate, but the ways they did so differed. For cyt-c, more than a monolayer was adsorbed onto the liposome surface prior to aggregation due to charge neutralization, whereas cyt c(552) caused aggregation at a protein/lipid ratio well below that required for charge neutralization. Therefore, although cyt-c may cause liposomes to aggregate by electrostatic interaction, cyt-c(552) does not act in this way. FTIR-attenuated total reflection spectroscopy (FTIR-ATR) revealed that cyt-c lost much of its secondary structure when bound to the hydrophobic surface of octadecyltrichlorosilane, whereas cyt-c(552) folds its domains into a beta-structure. This hydrophobic effect may be the key to the difference between the behaviors of the two cytochromes when bound to DOPG vesicles. 相似文献
11.
The extrinsic membrane protein cytochrome c binds to lipid mixtures containing negatively charged phospholipids such as diphosphatidylglycerol (DPG). In this study the effect of cytochrome c on the lipid distribution in a DPG-steroid spin-label (3-doxyl-5alpha-cholestane) model membrane system is examined. The electron spin resonance (ESR) line-shape changes indicate that cytochrome c induces lateral phase separation at room temperature. The resulting two-dimensional lipid distribution is nonrandom, consisting of clusters of phospholipids bound to cytochrome c and patches of steroid spin-label molecules. Phase separations are also observed in the three-component system: DPG, phosphatidylcholine, and 3-doxyl-5alpha-cholestane. 相似文献
12.
The interaction of cytochrome c with monolayers of phosphatidylethanolamine 总被引:5,自引:5,他引:0
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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. 相似文献
13.
Luiz C. Salay Thatyane M. Nobre Marcelle C. Colhone Maria E. D. Zaniquelli Pietro Ciancaglini Rodrigo G. Stabeli José Roberto S. A. Leite Valtencir Zucolotto 《Journal of peptide science》2011,17(10):700-707
This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA‐ KAAGQAALGAL‐NH2, DS 01) with phospholipid (PL) monolayers comprising (i) a lipid‐rich extract of Leishmania amazonensis (LRE‐La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid‐air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE‐La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE‐La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
14.
Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.
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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. 相似文献
15.
Hisactophilin-mediated binding of actin to lipid lamellae: a neutron reflectivity study of protein membrane coupling.
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C Naumann C Dietrich A Behrisch T Bayerl M Schleicher D Bucknall E Sackmann 《Biophysical journal》1996,71(2):811-823
The neutron reflectivity technique is applied to determine the adsorptive interaction of the 13.5-kDa actin-binding protein hisactophilin from Dictyostelium discoideum with lipid monolayers at a lateral pressure of 21 mN/m < or = pi < or = 25 mN/m at the air-water interface. We compare binding of natural hisactophilin exhibiting a myristic acid chain membrane anchor at the N-terminus (DIC-HIS) and a fatty acid-deficient genetic product expressed in Escherichia coli (EC-HIS). It is demonstrated that only the natural hisactophilin DIC-HIS is capable of mediating the strong binding of monomeric actin to the monolayer, where it forms a layer of about 40 A thickness corresponding to the average diameter of actin monomers. Monolayers composed of pure dimyristoyl phosphatidylcholine with fully deuterated hydrocarbon tails and headgroup (DMPC-d67) and 1:1 mixtures of this lipid with chain deuterated dimyristoyl phosphatidylglycerol (DMPG-d54) are studied on subphases consisting either of fully deuterated buffer (D2O) or of a 9:1 H2O/D2O buffer that matches the scattering length density of air (CMA buffer). The reflectivity data are analyzed in terms of layer models, consisting of one to three layers, depending on the contrast of the buffer and the system. We show that both protein species bind tightly to negatively charged 1:1 DMPC-d67/DMPG-d54 monolayers, thereby forming a thin and most probably monomolecular protein layer of 12-15 A thickness. We find that the natural protein (DIC-HIS) partially penetrates into the lipid monolayer, in contrast to chain-deficient species (EC-HIS), which forms only an adsorbed layer. The coverage of the monolayer with DIC-HIS strongly depends on the presence of anionic DMPG in the monolayer. At a bulk protein concentration of 1.5 micrograms/ml, the molar ratio of bound protein to lipid is about 1:45 for the 1:1 lipid mixture but only 1:420 for the pure DMPC. 相似文献
16.
Bailey A Zhukovsky M Gliozzi A Chernomordik LV 《Archives of biochemistry and biophysics》2005,439(2):211-221
The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells. 相似文献
17.
The interaction of cytochrome c with negatively charged lipids has been studied by resonance Raman spectroscopy of the protein heme group and 31P NMR of the phospholipid headgroups. The gel-to-fluid-phase transition of dimyristoylphosphatidylglycerol induces shifts in the conformational and coordination equilibria of the bound cytochrome c, as recorded by the resonance Raman spectra in the fingerprint and marker band regions. Conformational and coordination shifts of the bound cytochrome are also induced on admixture of dioleoylglycerol or dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol. In the case of dioleoylglycerol, significant changes take place even at levels as low as 5 mol %. Binding of cytochrome c induces or increases the content of near isotropically diffusing lipid registered by the 31P NMR spectra of the different lipids studied. Admixture of dioleoylglycerol also increases the bilayer curvature of dioleoylphosphatidylglycerol, inducing an inverted hexagonal phase at 50 mol % concentration; the tendency to spontaneous curvature in the lipid appears to relax the conformational change detected in the protein. 相似文献
18.
Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin 总被引:2,自引:1,他引:1
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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. 相似文献
19.
M. Montal 《生物化学与生物物理学报:生物膜》1973,298(3):750-754
Asymmetric lipid bilayers are formed by adjoining the hydrocarbon chains of two different lipid monolayers at the air-water interface through an aperture in a teflon partition separating two aqueous phases. It is shown that the addition of Ca2+ or polysine to the compartment limited by a monolayer of the neutral lipids glycerol dioleate or phosphatidylcholine results in no modification of the resistance and stability of the membrane, whereas a drastic decrease in both parameters is elicited by the presence of these ions on the opposite compartment containing a monolayer of the negatively charged cardiolipin or phosphatidylserine. The surface-charge dependence of this phenomenon indicates the persistence of the asymmetric lipid distribution in the bilayer after its formation from two different monolayers. 相似文献
20.
Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer. 相似文献