Protein charge determination and implications for interactions in cell extracts

Decades of dilute‐solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was ～2‐fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge‐neutral and “inert” in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge–charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed.     

Cytochrome c-Lipid Interactions: New Insights from Resonance Energy Transfer

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.     

Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study

Summary Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50–200 sec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10⁸ to 0.1 cm²), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential differenceV _c. For 1m alkali ion chloridesV _c was about 1 V. The temperature dependence of the electrical breakdown voltageV _c is comparable to that being observed with cell membranes.V _c decreases between 2 and 48°C from 1.5 to 0.6 V in the presence of 1m KCl.Breakdown experiments were also performed with lipid bilayer membranes composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability.The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).     

An imidoester spin probe of membrane protein interactions: application to cytochrome c

The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10^?9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10^?9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10^?9). Cytochrome c-oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.     

Rapid Nonsynonymous Evolution of the Iron-Sulfur Protein in Anthropoid Primates

Cytochrome c (CYC) and 9 of the 13 subunits of cytochrome c oxidase (complex IV; COX) were previously shown to have accelerated rates of nonsynonymous substitution in anthropoid primates. Cytochrome b, the mtDNA encoded subunit of ubiquinol-cytochrome c reductase (complex III), also showed an accelerated nonsynonymous substitution rate in anthropoid primates but rate information about the nuclear encoded subunits of complex III has been lacking.We now report that phylogenetic and relative rates analysis of a nuclear encoded catalytically active subunit of complex III, the ironsulfur protein (ISP), shows an accelerated rate of amino acid replacement similar to cytochrome b. Because both ISP and subunit 9, whose function is not directly related to electron transport, are produced by cleavage into two subunits of the initial translation product of a single gene, it is probable that these two subunits of complex III have essentially identical underlying rates of mutation. Nevertheless, we find that the catalytically active ISP has an accelerated rate of amino acid replacement in anthropoid primates whereas the catalytically inactive subunit 9 does not.     

Copper chaperones for cytochrome c oxidase and human disease

Biological processes in living cells are compartmentalized between lipid membranes. Integral membrane proteins often confer specific functions to these compartments and as such have a critical role in cellular metabolism and function. Cytochrome c oxidase is a macromolecular metalloprotein complex essential for the respiratory function of the cell. Elucidating the mechanisms of assembly of cytochrome c oxidase within the inner mitochondrial membrane represents a unique challenge for understanding metalloprotein biosynthesis. Elegant genetic experiments in yeast have defined several proteins required for copper delivery to cytochrome c oxidase. While the precise role of each of these proteins in copper incorporation remains unclear, recent studies have revealed that inherited mutations in two of these proteins can result in severe pathology in human infants in association with cytochrome c oxidase deficiency. Characterization of the molecular pathogenesis of these disorders offers new insights into the mechanisms of cellular copper metabolism and the role of these cytochrome c oxidase copper chaperones in human disease.     

Simultaneous measurements of optical and electrical properties of artificial membranes composed of mitochondrial lipids and their interaction with cytochromec

Summary A newly constructed cell, which allows simultaneous measurements of optical and electrical properties, was used to study bimolecular black membranes composed of beef heart mitochondrial lipids and their interaction with cytochromec.The results show that these highly charged membranes are stable only in relatively limited ranges of boundary conditions. In 0.1n KCl their maximum direct current (dc) resistance is 7×10⁸ Ohm cm²±10%; the series capacity at 1kHz is 0.43 F/cm²±3%; the entire thickness, determined by optical reflectivity, is 5.8±0.2 nm.The interaction between oxidized cytochromec and these lipid membranes is primarily of electrostatic nature, and dependent on the presence of highly charged phospholipids, such as diphosphatidyl glycerol (cardiolipin) and phosphatidyl ethanolamine. The attachment of cytochromec maximally causes a 2.5-fold increase in reflectivity, without any noticeable change in the capacity. This leads to a subsequent instability of the membrane (i.e., rupture) preceded by a rapid increase of the dc conductivity. This behavior is far less pronounced with reduced cytochromec.     

Interaction of cytochromec with phospholipid monolayers and bilayer membranes

Summary For the study of the interaction between oxidized cytochromec and phosphatidylinositide, two different model systems were used: (1) monolayers which were deposited after the method of Langmuir and Blodgett onto glass plates, and (2) bimolecular (“black”) membranes in aqueous phase. The amount of bound protein was determined with a sensitive spectrophotometer. It was found that at low ionic strength about 10¹³ cytochromec molecules per cm² are bound to the lipid surface, which nearly corresponds to a densely packed monolayer. At high ionic strength (∼ 0.1m) or low pH (pH<3), the adsorbed protein layer becomes unstable. This result indicates that the interaction is mainly electrostatic. In accordance with this conclusion is the observation that the rate of adsorption is diffusion controlled; i.e., almost every protein molecule hitting the surface is bound. The cytochromec monolayer can be reduced by ascorbate. In contrast to ferrocytochromec in solution, the bound ferrocytochrome was found to be autoxidable.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

Osmotically-induced and photo-induced deformations of disk membranes

Effects of the osmotic pressure of suspending medium on the size and shape of isolated bovine rod outer segment disk membranes in the unbleached and bleached states were studied by elastic and quasielastic light scattering in dilute solutions of aqueous sucrose (0-0.1 wt.%). Data for the translational diffusion coefficients, D, and the elastically scattered intensities were analyzed with use of the oblate ellipsoidal shell model, and the axial ratio, ρ, and the major semiaxis, b, of the ellipsoid were deduced as a function of the concentration, c, of sucrose for each photochemical state (unbleached and bleached) of the membranes. The unbleached and bleached membranes suspended in water (c = 0) were found to be spherical vesicles, i.e., ρ = 1, of 0.49 μm radius. As c was increased, however, ρ for the bleached membranes rather steeply increased approaching 3 at c = 0.1 wt.%, whereas that for the unbleached membranes gradually increased (up to 2 at c = 0.1%) after c exceeded 0.02%; at a fixed c, ρ increased upon bleaching. In all the cases, the deformation took place while b remained unchanged. The resulting contraction of the intravesicular volume induced either by the photo-bleaching or the osmotic pressure was accompanied by a significant decrease in the surface area of the membranes. The photo-induced contractions in the volume and the surface area amounted to 30–45% and 10–20%, respectively, in the concentration range from 0.02 to 0.1%. The dependence of D on c for each photochemical state was quantitatively explained by a simple model based on the Ca⁺ efflux from the intravesicular space to the bathing medium upon bleaching.     

The activity of the amphipathic peptide delta-lysin correlates with phospholipid acyl chain structure and bilayer elastic properties

Release of lipid vesicle content induced by the amphipathic peptide δ-lysin was investigated as a function of lipid acyl chain length and degree of unsaturation for a series of phosphatidylcholines. Dye efflux and peptide binding were examined for three homologous lipid series: di-monounsaturated, di-polyunsaturated, and asymmetric phosphatidylcholines, with one saturated and one monounsaturated acyl chain. Except for the third series, peptide activity correlated with the first moment of the lateral pressure profile, which is a function of lipid acyl chain structure. In vesicles composed of asymmetric phosphatidylcholines, peptide binding and dye efflux are enhanced compared to symmetric, unsaturated lipids with similar pressure profiles. We attribute this to the entropically more favorable interaction of δ-lysin with partially saturated phospholipids. We find that lipid acyl chain structure has a major impact on the activity of δ-lysin and is likely to be an important factor contributing to the target specificity of amphipathic peptides.     

Painted supported lipid membranes

We report herein measurements on a novel type of supported lipid films, which we call painted supported membranes (PSM). These membranes are formed in a self-assembly process on alkylated gold films from an organic solution. The formation process was investigated with surface plasmon resonance microscopy. The optical and electrical properties of the films were determined for various types of lipids and as a function of temperature by means of cyclic voltammetry and potential relaxation after charge injection. We could show that these films exhibit an extraordinarily high specific resistivity which, depending on the lipid, may be as high as 10⁹ ohm/cm². We could also show that due to this low conductivity, an electrical polarization across the PSM relaxes with characteristic time constants of up to 20 min. The electrical properties together with their high mechanical stability and accessibility to surface sensitive techniques make these films well suitable model membranes for optical and electrical investigations. Examples for such applications are given in the subsequent article by Seifert et al.     

Dendrimers complexed with HIV-1 peptides interact with liposomes and lipid monolayers

AimsWe have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD).MethodsInteraction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir–Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy.ResultsAll dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive.ConclusionsHIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.     

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin-cytochrome c interactions

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1-75 μM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK₂ decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.     

125I-UdR Induced Division Delay

The interaction of Ca++ with acidic phospholipids in black lipid films and lipid bilayers formed from two monolayers was studied by measuring their physical stability and conductance. It was found that the addition of CaCl2 to only one side of lipid bilayers formed from phosphatidylserine or cardiolipin does not appreciably change these parameters. In contrast, black films are unstable to the asymmetric addition of CaCl2. Therefore, the destabilizing effect of Ca++ cannot be attributed to a surface charge difference. The only variation in composition between both bilayer membranes, namely the solvent content of the bilayer, seems to be responsible for the distinctive effect of Ca++. A tentative explanation is presented.     

Purification and characterisation of periplasmic C-type cytochromes from Desulfovibrio desulfuricans (NCIMB 8372)

Periplasmic extract from Desulfovibrio desulfuricans (NCIMB 8372) was found to contain two different c-type cytochromes. One is tetraheme cytochrome c₃ and the other is monoheme cytochrome c₅₅₃. Cytochrome c₃ could be purified by a procedure involving only one chromatographic step, whereas cytochrome c₅₅₃ required several such steps. Cytochrome c₃ was found to have a relative molecular mass of 14300 and an isoionic point higher than 9. Analysis of the redox potentials indicated one heme at -260 mV and three hemes around -330 mV. Cytochrome c₅₅₃ had a relative molecular mass of 7200, an isoionic point higher than 9 and a redox potential of 0 mV.     

The role played by lipids unsaturation upon the membrane interaction of the Helicobacter pylori HP(2–20) antimicrobial peptide analogue HPA3

The HPA3 peptide is an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein, able to interact with zwitterionic lipid membranes and generate pores. Herein we focused on the importance of the degree of unsaturation of lipid acyl chains on HPA3 peptide-membrane interactions. Electrophysiology experiments carried out in reconstituted lipid membranes formed from phosphatidylcholines with one (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine − POPC) and two monounsaturated acyl chains (1,2-dioleoyl-sn-glycero-3-phosphocholine − DOPC) demonstrate that the lesser degree of the packing density of membrane lipids encountered in DOPC-based planar membranes greatly enhances the electric activity of pores created by the HPA3 peptide. Data derived from fluorescence spectroscopy experiments demonstrate that upon interaction with the bilayer, the HPA3 peptide translocates to the trans-side of the membrane. From the same experiments, we demonstrate that in the case of DOPC-based planar membranes, the net amount of HPA3 peptide which passes across the membrane and re-dissolves in the trans solution is almost 22% greater than POPC-based membranes. Such data further emphasize the modulatory role played by lipid acyl chain in determining antimicrobial peptides-lipids interactions, and demonstrate that small differences in unsaturation degree can impose a sizeable influence on HPA3 peptide activity.     

Regulation of fatty acid unsaturation in encystingAcanthamoeba cells

The degree of fatty acid unsaturation and average chain length are closely similar for microsomal membranes from exponential-phase trophozoites and cysts ofAcanthamoeba castellanii despite significant differences in fatty acid composition. The same trend was apparent for total fatty acids extracted from whole cells. The observations suggest that the organism regulates these lipid parameters during differentiation in order to maintain optimum membrane lipid viscosity, and are consistent with previous electron spin resonance measurements indicating that the fluidity of microsomal membranes does not change during encystment. About 75% of the microsomal fatty acids are unsaturated for both cysts and amoebae. Wide-angle X-ray diffraction of phospholipid liposomes prepared from lipid extracts of the membranes has indicted that this high level of unsaturation renders the phospholipid exclusively liquid-crystalline at temperatures as low as 9°C for rough microsomes and-1.5°C for smooth microsomes. Thus, by retaining a high proportion of unsaturated fatty acids throughout its differentiation cycle, the organism gains some protection in its natural soil habitat against lateral phase separation of membrane lipids.     

Spreading of biomembranes at the air/water interface1

This paper presents the compression isotherms obtained by spreading membranes of intestinal brush border, human erythrocyte and Escherichia coli (cytoplasmic) at the air/water interface. Unilamellar membrane films were formed, with a good yield, at zero surface pressure, whereas multilamellar structures were formed at high surface pressure. Once formed, the films were particularly stable and could be manipulated without any detectable loss. With doubly-labelled E. coli cytoplasmic membrane, we could show that phospholipids and proteins spread, with the same yield, as a single unit. Moreover, we studied the influence of hydrolytic enzymes, chemical agents and cations on the compression isotherm of biomembranes. The resultant change sin architecture of membrane films can provide a very simple method of studying the influence of membrane packing on catalytic activity and protein conformation of membrane-bound proteins.