The 5A structure of heterologously expressed plant aquaporin SoPIP2;1

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca²⁺-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg²⁺-dependent, Ca²⁺-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.     

Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles

Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the Rhodobacter sphaeroides light-harvesting 1 beta subunit in detergent micelles.

The light harvesting 1 antenna (LH1) complex from Rhodobacter sphaeroides funnels excitation energy to the photosynthetic reaction center. Our ultimate goal is to build up the structure of LH1 from structures of its individual subunits, much as the antenna can self-assemble from its components in membrane-mimicking detergent micelles. The beta subunit adopts a nativelike conformation in Zwittergent 3:12 micelles as demonstrated by its ability to take the first step of assembly, binding BChl a. Multidimensional NMR spectroscopy shows that the beta subunit folds as a helix((L12-S25))-hinge((G26-W28))-helix((L29-W44)) structure with the helical regions for the 10 lowest-energy structures having backbone rmsds of 0.26 and 0.24 A, respectively. Mn(2+) relaxation data and the protein-detergent NOE pattern show the C-terminal helix embedded in the micelle and the N-terminal helix lying along the detergent micelle surface with a 60 degrees angle between their long axes. (15)N relaxation data for residues L12-W44 are typical of a well-ordered protein with a correlation time of 8.25 +/- 2.1 ns. The presence of the hinge region placing the N-terminal helix along the membrane surface may be the structural feature responsible for the functional differences observed between the LH1 and LH2 beta subunits.     

Structural stability of the erythrocyte anion transporter, band 3, in native membranes and in detergent micelles.

The exothermic thermal denaturation transition of band 3, the anion transporter of the human erythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190-300 kcal mol-1) with an accompanying decrease in thermal denaturation temperatures (48-60 degrees C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75 degrees C) and DIDS-insensitive (62 degrees C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant differences was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C12E8) are required for optimal thermal stability of band 3.     

Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.     

Solution NMR spectroscopic characterization of human VDAC-2 in detergent micelles and lipid bilayer nanodiscs

Three isoforms of the human voltage-dependent anion channel (VDAC), located in the outer mitochondrial membrane, are crucial regulators of mitochondrial function. Numerous studies have been carried out to elucidate biochemical properties, as well as the three-dimensional structure of VDAC-1. However, functional and structural studies of VDAC-2 and VDAC-3 at atomic resolution are still scarce. VDAC-2 is highly similar to VDAC-1 in amino acid sequence, but has substantially different biochemical functions and expression profiles. Here, we report the reconstitution of functional VDAC-2 in lauryldimethylamine-oxide (LDAO) detergent micelles and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer nanodiscs. We find that VDAC-2 is properly folded in both membrane-mimicking systems and that structural and functional characterization by solution NMR spectroscopy is feasible. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

MD simulations of Mistic: conformational stability in detergent micelles and water

Mistic is an unusual membrane protein from Bacillus subtilis. It appears to fold and insert autonomously into a lipid bilayer and has been suggested as a tool that aids the targeting of eukaryotic membrane proteins to bacterial membranes. The NMR structure of Mistic in detergent (LDAO) micelles has revealed it to be a four alpha-helix bundle. From a structural perspective, Mistic does not resemble other membrane proteins. Its external surface is not very hydrophobic, and standard methods do not predict any of its helices to be in the transmembrane orientation. Molecular dynamics simulations (simulation times approximately 30 ns) in water and in detergent micelles have been used to explore the conformational stability of Mistic as a function of its environment. In water, the protein is stable, exhibiting no significant change in fold on a 30 ns time scale. In contrast, in three simulations in detergent micelles, the partial unfolding of Mistic occurred, whereby the H4 helix drifted away from the H1-H3 core. This was due to the penetration of detergent molecules between H4 and the remainder of the protein. This is unlike the behavior of several other membrane proteins, both alpha-helix bundles and beta-barrels, in comparable detergent micelle simulations. The unfolding of H4 from the H1-H3 core of Mistic could be partially reversed by a simulation in which the detergent molecules were removed, and the unfolded protein was simulated in water. These results suggest that Mistic may not be a stable integrated membrane protein but rather that it may undergo a conformational change upon interaction with a membrane or membrane-like environment.     

Isolation and lipid composition of spinach chloroplast envelope membranes

The quenching of the Chl a² fluorescence from spinach chloroplasts and chloroplast fragments by nitroaromatic compounds and the effect of added metal cations on the quenching rate is investigated. The extent of the quenching with nitrobenzene and 1,3-dinitrobenzene was found to be independent of whether Chl a is excited directly, or through Chl b by means of electronic energy transfer. On the basis of this, the contribution from a purely static mechanism is considered as unlikely.Nitroaromatics substituted with ionizable groups are almost equally effective quenchers for the fluorescence of Chl ain vivo and in methanol. On the other hand, nitroaromatics which are slightly soluble, or nearly insoluble, in water quench more strongly the fluorescence of Chl ain vivo. The overriding factor that determines the relation between the apparent and the true quenching constant appears to be the partition of the quencher in the lipid and the aqueous phases of the membrane suspension.Divalent metal cations enhance the quenching by nitrobenzene dramatically, most likely by increasing the hydrophobic character of the chloroplast membranes. This enhancement occurs at cation concentrations higher than those corresponding to the maximal turbidity increase of the membrane suspension; hence, it is attributed to ultrastructural changes of the membrane rather than to volume changes of the thylakoid. These changes may affect the extent of the quenching both by an increase in the local concentration of the nitroaromatic, and by an enhanced rate of excitation exchange among the chlorophylls.     

Structures of the FXYD regulatory proteins in lipid micelles and membranes

The FXYD membrane proteins constitute a family of conserved auxiliary subunits of the Na,K-ATPase, and have been the focus of recent attention due to their ability to finely regulate the activity of the enzyme complex in various physiological settings. In this review we describe the structures of the proteins, as well as their dynamics and their associations with the lipid bilayer membrane, which we have recently determined by NMR spectroscopy. Although the proteins are relatively small, their genes contain as many as six to nine small exons, and the coincidence of structured protein segments with their genetic elements suggests assembly from discrete structural modules through exon shuffling. The three-dimensional structures and backbone dynamics provide the foundation for understanding their intra-membrane association with the Na,K-ATPase α subunit, and the structure of FXYD1 suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the α subunit.     

Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: a comparative study of spinach SoPIP2;1 and E. coli AqpZ

This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.     

Effect of lead on the lipid metabolism in spinach leaves and thylakoid membranes

The effects of lead and sodium acetate treatment on the lipid composition of leaves, thylakoid membranes and cell debris of spinach were investigated. The concentration of lead in leaves and cell debris was higher than that in thylakoid membranes, probably due to a protection of photosynthetic apparatus. The lead treatment lead to decrease of contents of monogalactosyl diacylglycerols and phospholipids and to increase of the other glycolipids in the thylakoid membranes. There were no statistically significant differences between the total lipids of thylakoid membranes after incubation with lead and sodium acetate, which was an indication that in this case the effect of metal ion was not specific.     

Structure of the integrin beta3 transmembrane segment in phospholipid bicelles and detergent micelles

Integrin adhesion receptors transduce bidirectional signals across the plasma membrane, with the integrin transmembrane domains acting as conduits in this process. Here, we report the first high-resolution structure of an integrin transmembrane domain. To assess the influence of the membrane model system, structure determinations of the beta3 integrin transmembrane segment and flanking sequences were carried out in both phospholipid bicelles and detergent micelles. In bicelles, a 30-residue linear alpha-helix, encompassing residues I693-H772, is adopted, of which I693-I721 appear embedded in the hydrophobic bicelle core. This relatively long transmembrane helix implies a pronounced helix tilt within a typical lipid bilayer, which facilitates the snorkeling of K716's charged side chain out of the lipid core while simultaneously immersing hydrophobic L717-I721 in the membrane. A shortening of bicelle lipid hydrocarbon tails does not lead to the transfer of L717-I721 into the aqueous phase, suggesting that the reported embedding represents the preferred beta3 state. The nature of the lipid headgroup affected only the intracellular part of the transmembrane helix, indicating that an asymmetric lipid distribution is not required for studying the beta3 transmembrane segment. In the micelle, residues L717-I721 are also embedded but deviate from linear alpha-helical conformation in contrast to I693-K716, which closely resemble the bicelle structure.     

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.     

Heterodimerization of BAK and MCL-1 activated by detergent micelles

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.     

Structure and dynamics of the conserved protein GPI anchor core inserted into detergent micelles

A suitable approach which combines nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations have been used to study the structure and the dynamics of the glycosylphosphatidylinositol (GPI) anchor Manalphal-2Manalpha1-6Manalphal -4GlcNalpha1-6myo-inositol-1-OPO(3)-sn-1,2-dimyristoylglycerol (1) incorporated into dodecylphosphatidylcholine (DPC) micelles. The results have been compared to those previously obtained for the products obtainable from (1) after phospholipase cleavage, in aqueous solution. Relaxation and diffusion NMR experiments were used to establish the formation of stable aggregates and the insertion of (1) into the micelles. MD calculations were performed including explicit water, sodium and chloride ions and using the Particle Mesh Ewald approach for the evaluation of the electrostatic energy term. The MD predicted three dimensional structure and dynamics were substantiated by nuclear overhauser effect (NOE) measurements and relaxation data. The pseudopentasaccharide structure, which was not affected by incorporation of (1) into the micelle, showed a complex dynamic behaviour with a faster relative motion at the terminal mannopyranose unit and decreased mobility close to the micelle. This motion may be better described as an oscillation relative to the membrane rather than a folding event.     

Structure and thermodynamic properties of the complexes between phospholipase A2 and lipid micelles.

The interaction between porcine pancreatic phospholipase A2 and a homogeneous population of micelles of the subtrate analogue n-hexadecylphosphorylcholine containing 155 lipid monomers was studied by light scattering, equilibrium gel filtration, and isothermal calorimetry. From the detergent/protein molar ratio and the equivalent "molecular weight" of the resulting complex it is concluded that insertion of the enzyme into the detergent micelle results in a protein--detergent complex containing two phospholipase A2 molecules and 80 lipid monomers at 25 degrees C. The affinity constants and complex composition have been determined at different temperatures, allowing calculation of the thermodynamic parameters of the binding process. It is concluded that the interaction of phospholipase A2 with micellar lipids is predominantly hydrophobic.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.