Trasporto di materia in membrane polimeriche e nanocomposite per la separazione di gas

Membrane-based separation processes are acquiring, in the last years, an increasing importance because of their intrinsic energetic and environmental sustainability: some types of polymeric materials, showing adequate perm-selectivity features, appear rather suitable for these applications, because of their relatively low cost and easy processability. In this work have been studied two different types of polymeric membranes, in view of possible applications to the gas separation processes, i.e. Mixed Matrix Membranes (MMMs) and high free volume glassy polymers. Since the early 90’s, it has been understood that the performances of polymeric materials in the field of gas separations show an upper bound in terms of permeability and selectivity: in particular, an increase of permeability is often accompanied by a decrease of selectivity and vice-versa, while several inorganic materials, like zeolites or silica derivates, can overcome this limitation. As a consequence, it has been developed the idea of dispersing inorganic particles in polymeric matrices, in order to obtain membranes with improved perm-selectivity features. In particular, dispersing fumed silica nanoparticles in high free volume glassy polymers improves in all the cases gases and vapours permeability, while the selectivity may either increase or decrease, depending upon material and gas mixture: that effect is due to the capacity of nanoparticles to disrupt the local chain packing, increasing the dimensions of excess free volume elements trapped in the polymer matrix. In this work different kinds of MMMs were fabricated using amorphous Teflon® AF or PTMSP and fumed silica: in all the cases, a considerable increase of solubility, diffusivity and permeability of gases and vapours (n-alkanes, CO2, methanol) was observed, while the selectivity shows a non-monotonous trend with filler fraction. Moreover, the classical models for composites are not able to capture the increase of transport properties due to the silica addition, so it has been necessary to develop and validate an appropriate thermodynamic model that allows to predict correctly the mass transport features of MMMs. In this work, another material, called poly-trimethylsilyl-norbornene (PTMSN) was examined: it is a new generation high free volume glassy polymer that, like PTMSP, shows unusual high permeability and selectivity levels to the more condensable vapours. These two polymer differ each other because PTMSN shows a more pronounced chemical stability, due to its structure double-bond free. For this polymer, a set of Lattice Fluid parameters was estimated, making possible a comparison between experimental and theoretical solubility isotherms for hydrocarbons and alcoholic vapours: the successfully modelling task, based on application of NELF model, offers a reliable alternative to direct sorption measurement, which is extremely time-consuming due to the relevant relaxation phenomena showed by each sorption step. For this material also dilation experiments were performed, in order to quantify its dimensional stability in presence of large size, swelling vapours.

Numerical simulation of ion transport through ion channels and solid-state nanopores

Ion channels are pore-forming proteins that regulate the flow of ions across biological cell membranes. Ion channels are fundamental in generating and regulating the electrical activity of cells in the nervous system and the contraction of muscolar cells. Solid-state nanopores are nanometer-scale pores located in electrically insulating membranes. They can be adopted as detectors of specific molecules in electrolytic solutions. Permeation of ions from one electrolytic solution to another, through a protein channel or a synthetic pore is a process of considerable importance and realistic analysis of the main dependencies of ion current on the geometrical and compositional characteristics of these structures are highly required. The project described by this thesis is an effort to improve the understanding of ion channels by devising methods for computer simulation that can predict channel conductance from channel structure. This project describes theory, algorithms and implementation techniques used to develop a novel 3-D numerical simulator of ion channels and synthetic nanopores based on the Brownian Dynamics technique. This numerical simulator could represent a valid tool for the study of protein ion channel and synthetic nanopores, allowing to investigate at the atomic-level the complex electrostatic interactions that determine channel conductance and ion selectivity. Moreover it will provide insights on how parameters like temperature, applied voltage, and pore shape could influence ion translocation dynamics. Furthermore it will help making predictions of conductance of given channel structures and it will add information like electrostatic potential or ionic concentrations throughout the simulation domain helping the understanding of ion flow through membrane pores.

The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

Diffusion of laser polarized gases in MRI

The use of Magnetic Resonance Imaging (MRI) as a diagnostic tool is increasingly employing functional contrast agents to study or contrast entire mechanisms. Contrast agents in MRI can be classified in two categories. One type of contrast agents alters the NMR signal of the protons in its surrounding, e.g. lowers the T1 relaxation time. The other type enhances the Nuclear Magnetic Resonance (NMR) signal of specific nuclei. For hyperpolarized gases the NMR signal is improved up to several orders of magnitude. However, gases have a high diffusivity which strongly influences the NMR signal strength, hence the resolution and appearance of the images. The most interesting question in spatially resolved experiments is of course the achievable resolution and contrast by controlling the diffusivity of the gas. The influence of such diffusive processes scales with the diffusion coefficient, the strength of the magnetic field gradients and the timings used in the experiment. Diffusion may not only limit the MRI resolution, but also distort the line shape of MR images for samples, which contain boundaries or diffusion barriers within the sampled space. In addition, due to the large polarization in gaseous 3He and 129Xe, spin diffusion (different from particle diffusion) could play a role in MRI experiments. It is demonstrated that for low temperatures some corrections to the NMR measured diffusion coefficient have to be done, which depend on quantum exchange effects for indistinguishable particles. Physically, if these effects can not change the spin current, they can do it indirectly by modifying the velocity distribution of the different spin states separately, so that the subsequent collisions between atoms and therefore the diffusion coefficient can eventually be affected. A detailed study of the hyperpolarized gas diffusion coefficient is presented, demonstrating the absence of spin diffusion (different from particle diffusion) influence in MRI at clinical conditions. A novel procedure is proposed to control the diffusion coefficient of gases in MRI by admixture of inert buffer gases. The experimental measured diffusion agrees with theoretical simulations. Therefore, the molecular mass and concentration enter as additional parameters into the equations that describe structural contrast. This allows for setting a structural threshold up to which structures contribute to the image. For MRI of the lung this allows for images of very small structural elements (alveoli) only, or in the other extreme, all airways can be displayed with minimal signal loss due to diffusion.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

In vitro synthesis of the light-harvesting complex into artificial membrane systems

In green plants, the function of collecting solar energy for photosynthesis is fulfilled by a series of light-harvesting complexes (LHC). The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP), then imported into chloroplasts and assembled into photosynthetic thylakoid membranes. Knowledge about the regulation of the transport processes of LHCP is rather limited. Closely mimicking the in vivo situation, cell-free protein expression system is employed in this dissertation to study the reconstitution of LHCP into artificial membranes. The approach starts merely from the genetic information of the protein, so the difficult and time-consuming procedures of protein expression and purification can be avoided. The LHCP encoding gene from Pisum sativum was cloned into a cell-free compatible vector system and the protein was expressed in wheat germ extracts. Vesicles or pigment-containing vesicles were prepared with either synthetic lipid or purified plant leaf lipid to mimic cell membranes. LHCP was synthesized in wheat germ extract systems with or without supplemented lipids. The addition of either synthetic or purified plant leaf lipid was found to be beneficial to the general productivity of the expression system. The lipid membrane insertion of the LHCP was investigated by radioactive labelling, protease digestion, and centrifugation assays. The LHCP is partially protected against protease digestion; however the protection is independent from the supplemented lipids.

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.

The supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.

Origin and evolution of reactive and noble gases dissolved in matrix pore water

Reactive and noble gases dissolved in matrix pore water of low permeable crystalline bedrock were successfully extracted and characterized for the fist time based on drillcore samples from the Olkiluoto investigation site (SW Finland). Interaction between matrix pore water and fracture groundwater occurs predominately by diffusion. Changes in the chemical and isotopic composition of gases dissolved in fracture groundwater are transmitted and preserved in the pore water. Absolute concentrations, their ratios and the stable carbon isotope signature of hydrocarbon gases dissolved in pore water give valuable indications about the evolution of these gases in the nearby-flowing fracture groundwaters. Inert noble gases dissolved in matrix pore water and their isotopes combined with their in-situ production and accumulation rates deliver information about the residence time of pore water.

Redistribution of plasma membrane phosphatidylserine during erythroid development

Phosphatidylserine (PS) is not only one of the structural components of the plasma membrane, it also plays an important role in blood coagulation, and cell-cell interactions during aging and apoptosis.^ Here we studied some alterations that occur in membrane phosphatidylserine asymmetry during erythroid differentiation-associated apoptosis and erythrocyte aging and characterized some aspects in the regulation of PS asymmetry.^ Erythroleukemia cells, frequently used to study erythroid development, undergo apoptosis when induced to differentiate along the erythroid lineage. In the case of K562 cells induced to differentiate with hemin, this event is characterized by DNA fragmentation that correlates with downregulation of the survival protein BCL-xL and ultimately the result is cell death. We showed here that reorientation of PS from the inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase are also events observed upon hemin treatment. We observed that constitutive expression of BCL-2 did not inhibit the alterations caused by hemin in membrane lipid asymmetry and only slightly prevented hemin-induced DNA fragmentation. On the other hand, BCL-2 effectively inhibited actinomycin D and staurosporine-induced DNA fragmentation and the appearance of PS at the outer leaflet of these cells. z.VAD.fmk, a widely used caspases inhibitor, blocked DNA fragmentation induced by both hemin and actinomycin D but only inhibited PS externalization in cells treated with actinomycin D.^ These results showed that PS externalization occurs during differentiation-related apoptosis. Unlike the pharmacologically-induced event, however, hemin-induced PS redistribution seems to be regulated by a mechanism independent of BCL-2 and caspases.^ Membrane PS is externalized not only during apoptosis but also during red blood cell senescence. To study this event, we artificially induced cellular aging by in vitro storage or vesiculation in the presence of the amphipathic lipid dilauroylphosphatidylcholine. These cells were monitored for age-dependent changes in cell density by Percoll gradient centrifugation and assessed for alterations in membrane lipid asymmetry and their ability to be cleared in vivo. These experiments demonstrated a progressive increase in red cell density upon vesiculation and in vitro aging. The clearance rate of cells obtained after vesiculation, was biphasic in nature, showing a very rapid component together with a second component consistent with the clearance rates of control populations. Quantitation of PS in the outer leaflet of red cells revealed that membrane redistribution of PS occurred upon in vitro storage and vesiculation. Inhibition of the aminophospholipid translocase with the sulfhydryl-oxidant reagent pyridyldithioethylamine resulted in higher PS externalization and enhanced clearance of vesiculated RBC.^ These observations not only suggest that vesiculation may be the mechanism responsible for some of the characteristic changes in cell density and PS asymmetry that occur upon cell aging, but also confirm the role of PS in the recognition and clearance of senescent cells. ^

Rapid effects of 1alpha,25-dihydroxyvitamin D3 on osteoblasts: Studies of membrane transducers that affect calcium signals

1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] exerts pleiotropic effects on osteoblasts via both long-term nuclear receptor-mediated and rapid membrane-initiated pathways during bone remodeling and mineral homeostasis. This study explored the membrane transducers that mediate rapid effects of 1,25(OH)2D3 on osteoblasts, including sphingomyelinase (SMase) and L-type voltage sensitive calcium channels (VSCCs). ^ It was previously demonstrated that 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through VSCCs in ROS 17/2.8 osteoblasts, however the molecular identity of 1,25(OH)2D 3-regulated VSCC has not been known. In this study, on the basis of in vitro tests of three unique ribozymes specifically cleaving a1C mRNA, I transfected ROS 17/2.8 cells with vectors coding recombinant ribozyme modified with U1 snRNA structure, and successfully selected stable clonal cells in which the expression of a1C was strikingly reduced. Ca2+ influx studies in these cells compared to control transfectants showed selective attenuation of depolarization- and 1,25(OH)2D3-regulated Ca2+ responses. These results allow us to conclude that the cardiac ( a1C ) subtype of the L-type VSCC is the major membrane transducer of Ca 2+ influx in osteoblasts. ^ I also demonstrated that 1,25(OH)2D3 induces a rapid hydrolysis of membrane sphingomyelin (SM) in ROS 17/2.8 cells, with the concomitant generation of ceramide, detectable at 15 minute, and maximal at 1 hour after addition. Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC), downstream products of SM hydrolysis, but not ceramide, elicit Ca 2+ release from intracellular stores. Considering ceramide, sphingosine, and SPP as second messengers modulating intracellular kinases or phosphatases, these findings implicate sphingolipid-signaling pathways in transducing rapid effects of 1,25(OH)2D3 on osteoblasts. In structure/function analyses of sphingolipid signaling, it was observed that psychosine elicits Ca2+ release from intracellular stores. This challenges the dogma that sphingosine phosphorylation permits mobilization of Ca2+ , because psychosine is a sphingosine analog galactosylated at 1-carbon, preventing phosphorylation at that site. Psychosine is the pathological metabolite found in patients with Krabbe's disease, suggesting that psychosine disrupts the physiological sphingolipid signaling by chronic release of Ca2+ from intracellular stores. ^ Slower SM turnover than Ca2+ influx through VSCCs in response to 1,25(OH)2D3 demonstrates ceramide does not mediate the 1,25(OH)2D3-induced Ca2+ signaling, a conclusion endorsed further by the failure of ceramide to induce Ca 2+ signaling. ^

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Clinical outcomes following regenerative therapy of non-contained intrabony defects using a deproteinized bovine bone mineral combined with either enamel matrix derivative or collagen membrane

BACKGROUND The purpose of this study is to compare clinical outcomes in the treatment of deep non-contained intrabony defects (i.e., with ≥70% 1-wall component and a residual 2- to 3-wall component in the most apical part) using deproteinized bovine bone mineral (DBBM) combined with either enamel matrix protein derivative (EMD) or collagen membrane (CM). METHODS Forty patients with multiple intrabony defects were enrolled. Only one non-contained defect per patient with an intrabony depth ≥3 mm located in the interproximal area of single- and multirooted teeth was randomly assigned to the treatment with either EMD + DBBM (test: n = 20) or CM + DBBM (control: n = 20). At baseline and after 12 months, clinical parameters including probing depth (PD) and clinical attachment level (CAL) were recorded. The primary outcome variable was the change in CAL between baseline and 12 months. RESULTS At baseline, the intrabony component of the defects amounted to 6.1 ± 1.9 mm for EMD + DBBM and 6.0 ± 1.9 mm for CM + DBBM sites (P = 0.81). The mean CAL gain at sites treated with EMD + DBBM was not statistically significantly different (P = 0.82) compared with CM + DBBM (3.8 ± 1.5 versus 3.7 ± 1.2 mm). No statistically significant difference (P = 0.62) was observed comparing the frequency of CAL gain ≥4 mm between EMD + DBBM (60%) and CM + DBBM (50%) or comparing the frequency of residual PD ≥6 mm between EMD + DBBM (5%) and CM + DBBM (15%) (P = 0.21). CONCLUSION Within the limitations of the present study, regenerative therapy using either EMD + DBBM or CM + DBBM yielded comparable clinical outcomes in deep non-contained intrabony defects after 12 months.

Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents.

Purified membrane proteins are ternary complexes consisting of protein, lipid, and detergent. Information about the amounts of detergent and endogenous phospholipid molecules bound to purified membrane proteins is largely lacking. In this systematic study, three model membrane proteins of different oligomeric states were purified in nine different detergents at commonly used concentrations and characterized biochemically and biophysically. Detergent-binding capacities and phospholipid contents of the model proteins were determined and compared. The insights on ternary complexes obtained from the experimental results, when put into a general context, are summarized as follows. 1), The amount of detergent and 2) the amount of endogenous phospholipids bound to purified membrane proteins are dependent on the size of the hydrophobic lipid-accessible protein surface areas and the physicochemical properties of the detergents used. 3), The size of the detergent and lipid belt surrounding the hydrophobic lipid-accessible surface of purified membrane proteins can be tuned by the appropriate choice of detergent. 4), The detergents n-nonyl-β-D-glucopyranoside and Cymal-5 have exceptional delipidating effects on ternary complexes. 5), The types of endogenous phospholipids bound to membrane proteins can vary depending on the detergent used for solubilization and purification. 6), Furthermore, we demonstrate that size-exclusion chromatography can be a suitable method for estimating the molecular mass of ternary complexes. The findings presented suggest a strategy to control and tune the numbers of detergent and endogenous phospholipid molecules bound to membrane proteins. These two parameters are potentially important for the successul crystallization of membrane proteins for structure determination by crystallographic approaches.

SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.