Liposomal and Mitochondrial Cytochrome Oxidase Display Similar Bioenergetic Properties

Abstract

Cytochrome c oxidase, the terminal electron acceptor of the respiratory chain of mitochondria, is an integral membrane protein. The bioenergetic properties of cytochrome oxidase can be studied only when the macromolecule is inserted in a phospholipid bilayer, either in situ or after reconstitution into liposomal membranes. Reintegration of purified cytochrome oxidase in liposomes allows quantitative tests of mechanistic hypothesis concerning the functional properties of the enzyme. Small unilamellar vesicles are prepared by sonication of purified soybean asolectin, and reconstitution of cytochrome oxidase in the bilayer is carried out according to the cholate/dialysis procedure. The proteoliposomes are shown to mimick the mitochondrial state of the enzyme in so far as liposomal cytochrome oxidase : a) displays the same vectorial orientation, the cytochrome c binding site being externally exposed, b) pumps protons in the physiological inside/outside direction, and c) is functionally controlled by the transmembrane electrochemical gradient, i.e. displays respiratory control.     

Effects of Cerebroside and Cholesterol on the Reconstitution of Tonoplast H+-ATPase Purified from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

For an examination of the effects of cholesterol and cerebrosideon the rate and extent of proton-pumping across the membranesof proteoliposomes prepared with tonoplast H⁺-ATPase, the tonoplastH⁺-ATPase of mung bean (Vigna radiata L.) was purified by fastprotein liquid chromatography (FPLC) and incorporated into liposomesprepared from asolectin and cholesterol or cerebroside. Proteoliposomeswere formed after the removal of Triton X-100 from a mixtureof Triton X-100, asolectin and purified tonoplast H⁺-ATPaseby passage through an Ampure DT column. Proteoliposomes preparedfrom cholesterol and asolectin at a ratio of 45 : 55 (w/w) andat a ratio of lipid to protein of 200 : 1 (w/w) gave the largestpH gradient, as determined by the ATP-generated quenching ofquinacrine fluorescence. In the presence of cholesterol, thepH gradient formed across the membranes of proteoliposomes andthe average diameter of proteoliposomes increased about two-fold.The initial rate of proton-pumping decreased to 20% of thatobserved with proteoliposomes prepared from asolectin alone.The addition of cerebroside to asolectin at a ratio of 5 : 95(w/w) caused a 1.6-fold increase in the maximum pH gradientwithout any significant change in the initial rate of proton-pumpingor the diameter of proteoliposomes, but the maximum pH gradientdecreased greatly at ratios above 20 : 80 (w/w). The maximumpH gradient was transient and decreased spontaneously when onlyasolectin was used to prepare proteoliposomes, or when cerebrosideand asolectin were used together. Disappearance of the protongradient once it had formed and/or leakage of protons were suppressedby cholesterol at ratios above 30 : 70 (w/w). It was clear,therefore, that cholesterol and asolectin at ratios 30 : 70(w/w) to 45 : 55 (w/w) formed larger and more stable proteoliposomesthan did asolectin alone. ¹Present address: Laboratory of Climatic Stress Control, TohokuNational Agricultural Experiment Station, 4 Shimokuriyagawa,Morioka, Iwate, 020-01 Japan     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

Effect of different phospholipids on the reconstitution of two functions of the lactose carrier ofEscherichia coli

Summary The lactose carrier was extracted from membranes ofEscherichia coli and transport activity reconstituted in proteoliposomes containing different phospholipids. Two different assays f for carrier activity were utilized: counterflow and membrane potential-driven uptake. Proteoliposomes composed ofE. coli lipid or of 50% phosphatidylethanolamine–50% phosphatidylcholine showed very high transport activity with both assays. On the other hand, proteoliposomes containing asolectin, phosphatilcholine or 25% cholesterol/75% phosphatidylcholine showed good counterflow activity but poor membrane potentialdriven uptake. The discrepancy between the two types of transport activity in the latter group of three lipids is not due to leakiness to protons, size of proteoliposomes, or carrier protein content per proteoliposome. Apparently one function of the carrier molecule shows a broad tolerance for various phospholipids, while a second facet of the membrane protein activity requires very restricted lipid enviroment.     

Studies on the purified Na+,Mg(2+)-ATPase from Acholeplasma laidlawii B membranes: a differential scanning calorimetric study of the protein-phospholipid interactions

The purified Na+,Mg2(+)-ATPase from the Acholeplasma laidlawii B plasma membrane was reconstituted with dimyristoyl phosphatidylcholine and the lipid thermotropic phase behavior of the proteoliposomes formed was investigated by differential scanning calorimetry. The effect of this ATPase on the host lipid phase transition is markedly dependent on the amount of protein incorporated. At low protein/lipid ratios, the presence of increasing quantities of ATPase in the proteoliposomes increases the temperature and enthalpy while decreasing the cooperativity of the dimyristoyl phosphatidylcholine gel to liquid-crystalline phase transition. At higher protein/lipid ratios, the incorporation of increasing amounts of this enzyme does not further alter the temperature and cooperativity of the phospholipid chain-melting transition, but progressively and markedly decreases the transition enthalpy. Plots of lipid phase transition enthalpy versus protein concentration suggest that at the higher protein/lipid ratios each ATPase molecule removes approximately 1000 dimyristoyl phosphatidylcholine molecules from participation in the cooperative gel to liquid-crystalline phase transition of the bulk lipid phase. These results indicate that this integral transmembrane protein interacts in a complex, concentration-dependent manner with its host phospholipid and that such interactions involve both hydrophobic interactions with the lipid bilayer core and electrostatic interactions with the lipid polar head groups at the bilayer surface.     

Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.     

Biophysical and biochemical characterization of reconstituted and purified Rhodobacter sphaeroides cytochrome c oxidase in phospholipid vesicles sheds insight into its functional oligomeric structure

Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme.     

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.     

The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase.

Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.     

Reconstitution of cytochrome c oxidase in phospholipid vesicles containing polyvinylic polymers.

Cytochrome c oxidase was reconstituted in phospholipid vesicles in the presence of highly hydrophobic poly(vinyl alkanoate) polymers. Electron-microscopy observations demonstrated that polymer interaction with the lipid phase induces vesicles to adopt smaller diameters than those typical of standard proteoliposomes. Functional characterization of these polymer-proteoliposome structures indicates that the reconstitution of the enzyme proceeds efficiently without causing either scrambling of the protein orientation in the membrane or loss of respiratory control. A clear dependence of respiratory control ratio on vesicle size was also demonstrated, which is in agreement with a previous model proposed for control of activity of cytochrome c oxidase vesicles [Brunori, Sarti, Colosimo, Antonini, Malatesta, Jones & Wilson (1985) EMBO J. 4, 2365-2368].     

Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin

A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-100-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

A mechanism of respiratory control: studies with proteoliposomes containing cytochrome oxidase and bacteriorhodopsin

Both beef heart cytochrome oxidase and bacteriorhodopsin of Halobacterium halobium were reconstituted into liposomes by the sonication-cholate dialysis method. The proteoliposomes showed the respiratory control ratio of 4.2, and steady-state illumination of the vesicles lead to the 2.7-fold stimulation of the oxidase activity in the absence of uncouplers. The light-stimulated state 4 respiration increased with light intensity, but light had no effect on the oxidase activity that had been relieved by addition of uncouplers. Proteoliposomes with the photosensitive oxidase activity were also obtained when cytochrome oxidase vesicles were fused with bacteriorhodopsin vesicles in the presence of calcium chloride, and the extent of photoactivation was maximally 1.4-fold. The light-induced respiratory release was observed even in the presence of valinomycin or nigericin, indicating that the oxidase activity was sensitive to both the membrane potential and the pH gradient. We propose as a mechanism of the respiratory control that the process of proton transport to the reaction center for water formation is the rate limiting step for the cytochrome oxidase activity.     

Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation

Liposomes prepared by sonication of asolectin were fractionated by glycerol density gradient centrifugation, and the small liposomes contained in the upper region of the gradients were used for reconstitution of purified, radiolabeled Neurospora plasma membrane H+-ATPase molecules by our previously published procedures. The reconstituted liposomes were then subjected to two additional rounds of glycerol density gradient centrifugation, which separate the H+-ATPase-bearing proteoliposomes from ATPase-free liposomes by virtue of their greater density. The isolated H+-ATPase-bearing proteoliposomes in two such preparations exhibited a specific H+-ATPase activity of about 11 mumol of Pi liberated/mg of protein/min, which was approximately doubled in the presence of nigericin plus K+, indicating that a large percentage of the H+-ATPase molecules in both preparations were capable of generating a transmembrane protonic potential difference sufficient to impede further proton translocation. Importantly, quantitation of the number of 105,000-dalton ATPase monomers and liposomes in the same preparations by radioactivity determination and counting of negatively stained images in the electron microscope indicated ATPase monomer to liposome ratios of 0.97 and 1.06. Because every liposome in the preparations must have had at least one ATPase monomer, these ratios indicate that very few of the liposomes had more than one, and simple calculations show that the great majority of active ATPase molecules in the preparations must have been present as proton-translocating monomers. The results thus clearly demonstrate that 105,000-dalton monomers of the Neurospora plasma membrane H+-ATPase can catalyze efficient ATP hydrolysis-driven proton translocation.     

Topology of Cytochrome C Oxidase-Containing Proteoliposomes: Probes,Proteins and PH Gradients

Abstract

Cytochrome c oxidase-containing proteoliposomes (COV) prepared by cosonication show random orientation (45:55 in:out) of incorporated oxidase molecules; dialysed COV show 30:70 (in:out). Prepared COV show a pH gradient with an internal pH typically more acid than the medium. Such passive pH gradients probably reflect a Donnan distribution of anions such as chloride. The fluorescent pH probe 4-heptadecyl-7-hydroxycoumarin (HDHC) distributes between the two lipid leaflets at a ratio of between 30:70 and 33:67 (in:out) in cosonicated COV as measured by acid/base responses and quenching by p-xylene-b/s-pyridinium bromide. The HDHC pK was 8.25 in lauryl maltoside micelles, but membrane-bound HDHC showed a continuum of values ranging from 8.25 to 10.5. Maximum fluorescence in alkali was greater in lauryl maltoside than in COV. Active ΔpH gradients (alkaline inside) were generated by reductant and cytochrome c with aerobic oxidase-containing proteoliposomes ± valinomycin and nigericin. The gradients exceed 1.0 pH unit at low fluxes, higher than with water-soluble probes. ΔpH maintained between the bulk phases far from the membrane may be less than that at the lipid/water interface. With valinomycin (ΔΨ = 0), which accelerates ΔpH formation, ΔpH saturates at 1.0–1.2 units. Almost all the ΔΨ across the membrane can be converted into ΔpH by slow cation movement in the absence of ionophores. A gradient of either -90 mV (ΔΨ) or 1.0 pH unit (ΔpH) diminishes oxidase turnover by 80–90%. Control exerted by thermodynamically equivalent gradients is more effective with ΔpH than with ΔΨ. Differences between COV and mitochondria may be due to different rate-limiting electron transfer steps in the two systems.     

Influence of non-esterified fatty acids on respiratory control of reconstituted cytochrome-c oxidase

Bovine heart cytochrome-c oxidase was reconstituted in liposomes (asolectin) and the activity measured in the presence and absence of uncoupler at increasing concentrations of non-esterified fatty acids. Palmitic and stearic acids resulted in a decrease of about 40% in the respiratory control ratio at a concentration of 1 microM, when measured using a spectrophotometric procedure but not with a polarographic assay method. At higher fatty acid concentrations no further change was found. A 50% decrease in respiratory control was determined when the enzyme was reconstituted in pure phosphatidylcholine containing 2% cardiolipin. The respiratory control of reconstituted cytochrome-c oxidase from bovine liver was not influenced by fatty acids.     

Purified lac permease and cytochrome o oxidase are functional as monomers

Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination. The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas. In nonenergized proteoliposomes, both proteins appear to reconstitute as monomers based on (i) the variation of intramembrane particle density with protein concentration; (ii) the ratio of particles corresponding to each protein in proteoliposomes reconstituted with a known ratio of permease to oxidase; and (iii) the dimensions of the particles observed in tantalum replicas. The intramembrane particle diameters in tantalum replicas are about 20-25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of lac permease and cytochrome o. The diameters and heights of the permease and cytochrome o in tantalum replicas are 5.1 nm X 2.8 nm and 7.4 nm X 4.2 nm, respectively. Furthermore, a higher percentage of lac permease molecules exhibits a notch or cleft in tantalum replicas relative to platinum/carbon replicas. Importantly, the initial rate of lactose/H+ symport in proteoliposomes varies linearly with the ratio of lac permease to phospholipid, and no change is observed in either the size or distribution of lac permease molecules when the proteoliposomes are energized. The results taken as a whole provide a strong indication that both lac permease and cytochrome o reconstitute into proteoliposomes as monomers, that the permease does not dimerize in the presence of the H+ electrochemical gradient, and that both molecules are completely functional as monomers.     

Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions

The nicotinic acetylcholine receptor (nAChR) has been reconstituted in POPC vesicles at high lipid–protein (L/P) ratios for the preparation of supported lipid bilayers with a low protein density for studies of protein–lipid interactions using atomic force microscopy (AFM). Initial reconstitutions using a standard dialysis method with bulk L/P ratios ranging from 20:1 to 100:1 (w/w) gave heterogeneous samples that contained both empty vesicles and proteoliposomes with a range of L/P ratios. This is problematic because empty vesicles adsorb and rupture to form bilayer patches more rapidly than do protein-rich vesicles, resulting in the loss of protein during sample washing. Although it was not possible to find reconstitution conditions that gave homogeneous populations of vesicles with high L/P ratios, an additional freeze–thaw cycle immediately after dialysis did reproducibly yield a fraction of proteoliposomes with L/P ratios above 100:1. These proteoliposomes were separated by sucrose gradient centrifugation and used to prepare supported bilayers with well-separated individual receptors and minimal adsorbed proteoliposomes. AFM images of such samples showed many small features protruding from the bilayer surface. These features range in height from 1 to 5 nm, consistent with the smaller intracellular domain of the protein exposed, and have lateral dimensions consistent with an individual receptor. Some bilayers with reconstituted protein also had a small fraction of higher features that are assigned to nAChR with the larger extracellular domain exposed and showed evidence for aggregation to give dimers or small oligomers. This work demonstrates the importance of using highly purified reconstituted membranes with uniform lipid–protein ratios for AFM studies of integral membrane protein–lipid interactions.