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Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.

The study of biomimetic model membrane systems undergoing liquid-ordered (Lo)-liquid-disordered (Ld) phase separation using spectroscopic methods has played an important role in understanding the properties of lipid rafts in plasma membranes. In particular, the membrane-associated fluorescence probe Laurdan has proved to be a very efficient reporter of Lo-Ld phase separation in lipid bilayers using the general polarization (GP) parameter. A limitation of the GP approach is that it monitors only global average packing so that the contribution of each phase remains undetermined. The decomposition of Laurdan emission spectra has been proposed as an additional approach to overcoming this limitation. Here, further developments of this method for the study of Lo-Ld phase separation are described here for Laurdan in sphingomyelin-phosphatidylcholine-cholesterol large unilamellar vesicles. Lipid compositions corresponding to homogeneous Lo or Ld phases as well as undergoing thermally induced Lo-Ld phase separation were investigated. In addition, the occurrence of phase separation was checked by the fluorescence imaging of giant unilamellar vesicles. Decomposition into three log-normal components is used to show that an intermediate energy component is specifically associated with the occurrence of the Lo phase, with a small contribution from this component occurring above the phase-separation temperature being attributable to phase fluctuations. The ratio R X of the relative area of this intermediate-energy peak to that of the low-energy peak is shown to provide a straightforward index of Lo-Ld phase separation as a function of temperature, which is occasionally more sensitive than GP. It is also shown that R X can be used in conjunction with GP to gain further insight into Lo-Ld, the phase-separation processes. This latter feature is illustrated by the influence of the alcohol butanol on the Lo-Ld phase separation in sphingomyelin-phosphatidylcholine-cholesterol bilayers by showing that the effect of the alcohol occurs specifically at the onset of the phase separation, indicating a line tension mechanism. It is proposed that the three components of log-normal decomposition approaching Laurdan emission spectra provide a useful improvement for characterizing Lo-Ld phase-separation phenomena.

Alzheimer's disease (AD) is characterized by the overproduction of the amyloid-β peptide (Aβ) which forms fibrils under the influence of raft microdomains containing the ganglioside GM1. Raft-mimicking artificial liquid ordered (Lo) domains containing GM1 enhance amyloid-β polymerization. Other experiments suggest that Aβ binds preferably to the non-raft liquid disordered (Ld) phase rather than to the Lo phase in the presence of GM1. Here, the interaction of Aβ(1-42) with GM1-containing biphasic Lo-Ld giant vesicles was investigated. Fluorescence colocalisation experiments confirm that Aβ(1-42) binds preferentially to the Ld phase. The effect of Aβ(1-42) on the Lo-Ld size dynamics was studied using photoinduced spinodal decomposition which mimics the nanodomain-microdomain raft coalescence. Aβ affects the kinetics of the coarsening phase and the size of the resulting microdomains. The effect depends on which phase is in a majority: when the Lo microdomains are formed inside an Ld phase, their growth rate becomes slower and their final size smaller in the presence of Aβ(1-42), whereas when the Ld microdomains are formed inside an Lo phase, the growth rate becomes faster and the final size larger. Fluorimetric measurements on large vesicles using the probe Laurdan indicate that Aβ(1-42) binding respectively increases or decreases the packing of the Ld phase in the presence or absence of GM1. The differential effects of Aβ on spinodal decomposition are accordingly interpreted as resulting from distinct effects of the peptide on the Lo-Ld line tension modulated by GM1. Such modulating effect of Aβ on domain dynamics could be important for lipid rafts in signaling disorders in AD as well as in Aβ fibrillation.

Most biological molecules contain acido-basic groups that modulate their structure and interactions. A consequence is that pH gradients, local heterogeneities and dynamic variations are used by cells and organisms to drive or regulate specific biological functions including energetic metabolism, vesicular traffic, migration and spatial patterning of tissues in development. While the direct or regulatory role of pH in protein function is well documented, the role of hydrogen and hydroxyl ions in modulating the properties of lipid assemblies such as bilayer membranes is only beginning to be understood. Here, we review approaches using artificial lipid vesicles that have been instrumental in providing an understanding of the influence of pH gradients and local variations on membrane vectorial motional processes: migration, membrane curvature effects promoting global or local deformations, crowding generation by segregative polarization processes. In the case of pH induced local deformations, an extensive theoretical framework is given and an application to a specific biological issue, namely the structure and stability of mitochondrial cristae, is described. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A recurring question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, potential effects of these bioactive lipids on physicochemical properties of the lipid bilayer of cell membranes were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or, for the first time, together, in large or giant phosphadidylcholine (PC) unilamellar vesicles. Three bilayer properties were examined: membrane surface charge, lipid packing, and formation of membrane microdomains. Sph and S1P appear to have distinct, when not inverse, effects on all three properties. Besides, when both sphingolipids are mixed together, their effects on lipid packing are synergistic, whereas their effects on microdomain formation and zeta-potential are mostly antagonistic. These results are interpreted as arising from different electrostatic interactions between lipid headgroups. In particular, Sph and S1P may interact together electrostatically and form a complex. These mostly inverse and opposing effects of both single-chain phospholipids on membrane physical properties might be involved in their antagonistic role in regulating cell fate. Particularly, the mutual interaction between Sph and S1P as a complex might be able to sequester both molecules in a biologically inactive form and therefore to promote a mutual regulation of their biological activities, depending on their ratio, consistent with the sphingolipid rheostat.

In a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is attributable to direct phospholipid packing modification in the outer leaflet, that is promoted by protonation of cardiolipin headgroups. In this study, we compare the case of cardiolipin-containing giant unilamellar vesicles with that of giant unilamellar vesicles that contain phosphatidylglycerol (PG). Local acidification also promotes formation of tubules in the latter. However, compared with cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling, and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH gradient. Simulations using a fully nonlinear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondrial activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondrial function., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting "raft-like" Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A "morphological" phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, "arresting" domain growth and thereby stabilizing Lo nanodomains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells.

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles., (© 2011 American Chemical Society)

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.

Several preclinical and clinical studies suggest the importance of naturally occurring polymorphisms of drug transporters in the individual difference of drug response. To functionally validate the nonsynonymous polymorphisms of ABCB1 (P-glycoprotein/MDR1) in vitro, we generated SNP variant forms (i.e., S400N, R492C, R669C, I849M, A893P, A893S, A893T, M986V, A999T, P1051A, and G1063A) and expressed them in Sf9 cells. The kinetic properties (Km and Vmax) of those variants were analyzed by measuring the ATPase activity to obtain the ATPase profile for each variant toward structurally unrelated substrates. On the basis of the experimental data, we determined the substrate specificity of ABCB1 WT and its variants by the quantitative structure-activity relationship (QSAR) analysis method. While several SNP variants appeared to influence the substrate specificity of ABCB1, the nonsynonymous polymorphisms of 2677G > T, A, or C at amino acid position 893 (Ala > Ser, Thr, or Pro) have great impacts on both the activity and the substrate specificity of ABCB1. The A893P variant (2677G > C), a rare mutation, exhibited markedly high activity of ATPase toward different test compounds. Molecular dynamics (MD) simulation based on a three-dimensional structural model of human ABCB1 revealed that multiple kinks are formed in the intracellular loop between transmembrane domains 10 and 11 of the A893P variant (2677G > C) protein. The polymorphisms of 2677G, 2677T, and 2677A exhibit wide ethnic differences in the allele frequency, and these nonsynonymous polymorphisms are suggested to be clinically important because of their altered ATPase activity and substrate specificity toward different drugs.

Tetraspanins are a superfamily of transmembrane proteins implicated in cellular development, motility, and activation through their interactions with a large range of proteins and with specific membrane microdomains. The complete three-dimensional structure of the tetraspanin CD81 has been predicted by molecular modeling and from the crystallographic structure of the EC2 large extracellular domain. Periodicity of sequence conservation, homology modeling, secondary structure prediction, and protein docking were used. The transmembrane domain appears organized as a four-stranded left-handed coiled coil directly connecting to two helices of the EC2. A smaller extracellular loop EC1 contains a small largely hydrophobic beta-strand that packs in a conserved hydrophobic groove of the EC2. The palmitoylable intracellular N-terminal segment forms an amphipathic membrane-parallel helix. Structural variability occurs mainly in an hypervariable subdomain of the EC2 and in intracellular regions. Therefore, the variable interaction selectivity of tetraspanins originates both from sequence variability within structurally conserved domains and from the occurrence of small structurally variable domains. In CD81 and other tetraspanins, the numerous membrane-exposed aromatic residues are asymmetrically clustered and protrude on one side of the transmembrane domain. This may represent a functional specialization of these two sides for interactions with cholesterol, proteins, or membrane microdomains.

The effect of detergents on giant unilamellar vesicles (GUVs) composed of phosphatidylcholine, sphingomyelin and cholesterol and containing liquid-ordered phase (l(o)) domains was investigated. Such domains have been used as models for the lipid rafts present in biological membranes. The studied detergents included lyso-phosphatidylcholine, the product of phospholipase A2 activity, as well as Triton X-100 and Brij 98, i.e. detergents used to isolate lipid rafts as DRMs. Local external injection of each of the three detergents at subsolubilizing amounts promoted exclusion of l(o) domains from the GUV as small vesicles. The budding and fission processes associated with this vesiculation were interpreted as due to two distinct effects of the detergent. In this framework, the budding is caused by the initial incorporation of the detergent in the outer membrane leaflet which increases the spontaneous curvature of the bilayer. The fission is related to the inverted-cone molecular shape of the detergent which stabilizes positively curved structures, e.g. pores involved in vesicle separation. On the other hand, we observed in GUVs neither domain formation nor domain coalescence to be induced by the addition of detergents. This supports the idea that isolation of DRM from biological membranes by detergent-induced extraction is not an artifact. It is also suggested that the physico-chemical mechanisms involved in l(o) domain budding and fission might play a role in rafts-dependant endocytosis in cells.

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.

T-cell activation is initiated by the concerted engagement of the T-cell receptor and different co-stimulatory molecules, and requires cytoskeleton-dependent membrane dynamics. Here, we have studied the relationships between tetraspanins, cytoskeleton and raft microdomains, and their relevance in T-cell signaling. Localization studies and density-gradient flotation experiments indicate that part of tetraspanins localizes in raft microdomains linked to the actin cytoskeleton. First, partial coalescence of lipid raft is triggered by tetraspanin cross-linking and results in large caps in which F-actin also concentrates. Second, the amount of tetraspanins, which are recovered in the cholesterol-dependent insoluble fractions of low and intermediate density, and which appears to be membrane vesicles by electron microscopy, is under cytoskeletal influence. Disruption of actin filaments enhances the amount of tetraspanins recovered in typical raft fractions, whereas F-actin-stabilizing agents induce the opposite effect. Our data also reveal that CD82 constitutes a link between raft domains and the actin cytoskeleton, which is functionally relevant. First, tetraspanin signaling induces a selective translocation of CD82 from detergent-resistant membrane fractions to the cytoskeleton-associated pellet. Second, all functional effects linked to CD82 engagement, such as adhesion to culture plates, formation of actin bundles and early events of tyrosine phosphorylation, are abolished, or strongly reduced, by cholesterol depletion. We also show that dynamic relocalization of CD82 and F-actin at the periphery of the immune synapse is induced upon contact of T cells with antigen-presenting cells. This suggests that the tetraspanin web might participate in the membrane dynamics required for proper T-cell signaling. More generally, the interaction of tetraspanins with raft domains and with the actin cytoskeleton might relate with their role in many cellular functions as membrane organizers.

The validity of the structure of the Escherichia coli MsbA lipid transporter as a model from the mdr1 P-glycoprotein has been evaluated. Comparative sequence analyses, motif search and secondary structure prediction indicated that each of the two P-glycoprotein halves is structurally similar to the MsbA monomer and also suggested that the open dimer structure is valid for P-glycoprotein. Homology modeling was used to predict the structure of P-glycoprotein using MsbA as a template. The resulting modeled structure allowed a detailed study of the interactions between the intracellular domain and the nucleotide binding domain and suggested that these contacts are involved in mediating the coupling between nucleotide binding domain conformational changes and transmembrane helices reorientation during transport. In P-glycoprotein, the internal chamber open to the inner leaflet and the inner medium is significantly different in size and charge than in MsbA. These differences can be related to those of the transported substrates. Moreover an ensemble of 20 conserved aromatic residues appears to border the periphery of each side of the chamber in P-glycoprotein. These may be important for size selection and proper positioning of drugs for transport. The relevance of the modeled conformation to P-gp function is discussed.

The transbilayer movement of glycosphingolipids has been characterized in Golgi, ER, plasma, and model membranes using spin-labeled and fluorescent analogues of the monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid lactosylceramide. In large unilamellar lipid vesicles, monohexosylsphingolipids underwent a slow transbilayer diffusion (half-time between 2 and 5 h at 20 degrees C). Similarly, the inward redistribution of these sphingolipids in the plasma membrane of the hepatocyte-like cell line HepG2 and of erythrocytes was slow. However, in rat liver ER and Golgi membranes, we found a rapid transbilayer movement of spin-labeled monohexosylsphingolipids (half-time of approximately 3 min at 20 degrees C), which suggests the existence of a monohexosylsphingolipid flippase. The transbilayer movement of glucosylceramide in the Golgi and the ER displayed a saturable behavior, was inhibited by proteolysis, did not require Mg-ATP, and occurs in both directions. Treatment with DIDS inhibited the flip-flop of glucosylceramide but not that of phosphatidylcholine. These data suggest that the transbilayer movement of monoglucosylceramide in the ER and in the Golgi involves a protein that could be distinct from that previously evidenced for glycerophospholipids in the ER. In vivo, transbilayer diffusion should promote a symmetric distribution of monohexosylsphingolipids which are synthesized in the cytosolic leaflet. This should allow glucosylceramide rapid access to the lumenal leaflet where it is converted to lactosylceramide. No significant transbilayer movement of lactosylceramide occurred in both artificial and natural membranes over 1 h. Thus, lactosylceramide, in turn, is unable to diffuse to the cytosolic leaflet and remains at the lumenal leaflet where it undergoes the subsequent glycosylations.

The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold.

The present work evaluates the use of intermolecular polypeptide-detergent 1H through-space connectivities to determine the bilayer exposed-surface and the bilayer topography of membrane polypeptides solubilized in non- deuterated detergents. For this purpose, the membrane peptide gramicidin A, solubilized in non-deuterated sodium dodecylsulfate as its dimeric β6,3 helix channel conformation was used. For this peptide, a high-resolution 3D structure, as well as reasonable assumptions concerning its membrane arrangement, exist. Band-selective 2D NOESY, ROESY and 3D NOESY-NOESY experiments were used to detect detergent-polypeptide through-space correlations in the presence of an excess of the non-deuterated detergent. The observed intermolecular NOEs appear to be strongly temperature- dependent. Based on the known 3D structure of the gramicidin channel, the detergent-polypeptide through-space correlations appear to be selective for 1H located on the hydrophobic surface of gramicidin A with very few contributions from interior 1H or water-exposed 1H. It is suggested that this method can be of general use to evaluate the bilayer-exposed surface and topography of membrane peptides and small proteins.

Puroindoline-a is the main component of a new family of proteins that has been suggested to exert an antimicrobial activity in plant seeds through an interaction with lipid membranes. Here the interaction of puroindoline-a with model phospholipid membranes and micelles has been studied using intrinsic tryptophan fluorescence, fluorescence polarization of diphenyl hexatriene, and proteolysis experiments. The protein appears to interact with both zwitterionic and negative phospholipids. The interaction with phosphatidylcholine is characterized by low-affinity surface binding with very limited penetration into the hydrophobic membrane interior. On the other hand, the interaction with phosphatidylglycerol displays a high affinity and involves a partial penetration of the protein into the bilayer interior that disrupts acyl chain packing. The specificity appears to be due to the presence of a stretch of positively charged residues in the protein sequence. In all, the lipid-binding properties of puroindoline-a resemble those of cardiotoxins, another family of proteins for which a disruptive effect on the membrane structure has been involved to explain their biological function., (Copyright 1998 Academic Press.)

Recently the use of band-selective excitation to obtain 1H 2D NMR spectra of membrane peptides and proteins in non-deuterated detergents has been demonstrated [Seigneuret, M. and Levy, D. (1995) J. Biomol. NMR, 5, 345-352]. A limitation of the method was the inability to obtain through-space correlation between aliphatic protons. Here, a 3D F3-band-selective NOESY-TOCSY experiment is described that allows such correlations to be observed in the presence of an excess of non-deuterated detergent. Application to the measurement of proximities between aliphatic protons of the membrane peptide mastoparan X solubilized in non-deuterated n-octylglucoside is presented. With this additional experiment, it is now possible to obtain the same amount of structural constraints on membrane peptides and protein in non-deuterated detergent as in deuterated detergent and therefore to perform complete structural studies.

Application of 1H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a 1H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by 1H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the 1H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein 1H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and alpha-protons. Regioselective COSY spectra provide scalar coupling constants between amide and alpha-protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution 13C-NMR experiments have been performed on bacteriorhodopsin biosynthetically labeled with carbonyl-13C amino acids and solubilized in the detergent dodecylmaltoside. 13C-NMR spectra showing good resolution were obtained in the case of labeled amino acids moderately represented in the BR sequence. For BR labeled with [13C]carbonyl methionine, several sequence-specific assignment could be performed by co-labeling with 15N amino acids or proteolysis. These assignments were used to obtain structural data on BR. Water-exposure of methionine side chains in the protein was assessed by studying, using NMR, their oxidation by hydrogen peroxide. Local secondary structure at the level of methionine residues was monitored through the effect of 1H-2H exchange on NMR spectra. It was concluded that Met32, Met68 and Met163 are peripheral while all 6 other methionine residues are deeply embedded within hydrophobic alpha-helices. These results confirm the current model of the BR folding and secondary structure.

The proton transport membrane protein bacteriorhodopsin has been biosynthetically labeled with [methyl-13C]methionine and studied by high-resolution 13C NMR after solubilization in the detergent Triton X-100. The nine methionine residues of bacteriorhodopsin give rise to four well-resolved 13C resonances, two of which are shifted upfield or downfield due to nearby aromatic residues. Methionine residues located on the hydrophilic surfaces, on the hydrophobic surface, and in the interior of the protein could be discriminated by studying the effects of papain proteolysis, glycerol-induced viscosity increase, and paramagnetic broadening by spin-labels on NMR spectra. Such data were used to evaluate current models of the bacteriorhodopsin transmembrane folding and tertiary structure. T2 and NOE measurements were performed to study the local dynamics of methionine residues in bacteriorhodopsin. For the detergent-solubilized protein, hydrophilic and hydrophobic external residues undergo a relatively large extent of side chain wobbling motion while most internal residues are less mobile. In the native purple membrane and in reconstituted bacteriorhodopsin liposomes, almost all methionine residues have their wobbling motion severely restricted, indicating a large effect of the membrane environment on the protein internal dynamics.

The processes of liposome solubilization and reconstitution were studied by using n-dodecyl octaethylene glycol monoether (C12E8). The solubilization of large unilamellar liposomes prepared by reverse-phase evaporation was systematically investigated by turbidity, 31P nuclear magnetic resonance, and centrifugation experiments. The solubilization process is well described by the three-stage model previously proposed for other detergents, and our results further demonstrate the validity of some of the postulates related to this model. In stage I, the detergent distributes between the bilayers and the aqueous solution with a partition coefficient of 1.6 mM-1. In stage II, the detergent-saturated liposomes convert into mixed micelles, the conversion being complete by stage III where all the phospholipids are present as mixed micelles. The agreement between the three methods was excellent, and the results allowed quantitative determination of the effective detergent to phospholipid ratios at which the lamellar to micellar transformation begins and is complete, which amounted to 0.66 and 2.2 (mol/mol), respectively. Furthermore, compositional analysis determined from centrifugation experiments directly demonstrate that the properties of detergent-saturated liposomes and mixed micelles remain constant throughout most of stage II: the C12E8 to phospholipid ratios in the pelleted vesicles and in micelles are constant during stage II and similar to the ratios at which stage II was initiated and complete, respectively. On the other hand, bilayer formation upon detergent removal from mixed C12E8-phospholipid micelles by SM2 Bio-Beads is demonstrated to be the symmetrical opposite of bilayer solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)

This investigation uses a system for inducing and detecting anti-tetanus toxoid antibody (anti-TT) synthesis to study specific antibody (Ab) synthesis by bone marrow mononuclear cells (MC). We measured the amounts of anti-TT secreted and the number of B cells secreting antibody (Ab). The ELISA plaque detects single B cells secreting specific Ab. The results show that (1) spontaneous anti-TT secretion by MC is higher than spontaneous anti-TT secretion by peripheral blood lymphocytes (PBL) using an ELISA plaque (P less than 0.01); (2) spontaneous anti-TT production by MC correlated with the serum anti-TT titers as measured by an ELISA (r = 0.75, P = 0.005); (3) two types of marrow B cells were identified--one that spontaneously secretes anti-TT and another that produces anti-TT after TT-stimulation; (4) the frequency of anti-TT-secreting B cells is higher in MC than in PBL; (5) the amount of Ab secreted per marrow B cell is not different from that secreted by a peripheral B cell; and (6) marrow B cells could be induced to produce anti-TT in vitro up to 10 months without added cytokines. These results show that bone marrow is a major repository for differentiated B cells that spontaneously produce Abs to maintain circulating Abs titers and for memory B cells that can be induced to produce specific Ab.

Equilibrium and kinetic aspects of Triton X-100 adsorption onto hydrophobic Bio-Beads SM2 were investigated in detail using the batch procedure originally described by Holloway, P.W. (1973) Anal. Biochem. 53, 304-308. The results demonstrated the importance of the initial detergent concentration, the amount of beads, the commercial source of the detergent, the temperature and the presence of phospholipids in determining the rates of Triton X-100 adsorption onto Bio-Beads. One of the main findings was that Bio-Beads allowed the almost complete removal of Triton X-100, whatever the initial experimental conditions. It was shown that monomeric as well as micellar detergent could be adsorbed and that a key factor in determining the rate of detergent removal was the availability of the free bead surface. Rates of detergent removal were found to be linearly related to the amount of beads even for bead concentrations above those sufficient to remove all the detergent initially present. Adsorptive capacity of phospholipids onto Bio-Beads SM2 was also analyzed and found to be much smaller (2 mg lipid per g of wet beads) than that of Triton X-100 (185 mg TX 100 per g of wet beads). A more general aspect of this work was that the use of Bio-Beads SM2 provided a convenient way for varying and controlling the time course of Triton X-100 removal. The method was further extended to the formation of liposomes from phospholipid-Triton X-100 micelles and the size of the liposomes was found to be critically dependent upon the rate of detergent removal. A general procedure was described to prepare homogeneous populations of vesicles. Freeze-fracture electron microscopy and permeability studies indicated that the liposomes thus obtained were unilamellar, relatively large and impermeable. Noteworthy, this new procedure was shown to be well suited for the reconstitution of different membrane transport proteins such as bacteriorhodopsin, Ca2(+)-ATPase and H(+)-ATPase.

The rise in rates of infection in adults over the age of 60 is accompanied by a decreased ability of older adults to make specific immune responses after immunization with a variety of specific antigens (Ag). This investigation delineates age-related changes in Ag-specific humoral immunity, comparing adults over age 60 to young adults aged 18-40, using tetanus toxoid (TT) as an immunologic probe. A culture system which does not require TT booster immunizations of study subjects was used to induce in vitro specific antibody responses. The amount of anti-TT antibody (Ab) produced in serum and in culture was measured by a TT-specific enzyme-linked immunosorbent assay (ELISA). The numbers of anti-TT Ab-secreting B cells were measured by a TT-specific ELISA-plaque assay. The TT-specific responses of old subjects were significantly less than that seen for young control subjects in the following measures: (1) serum anti-TT Ab titers (mean +/- S.E. log 2 titer = 3.3 +/- 1.1 vs. 9.5 +/- 1.4, P less than 0.01); (2) anti-TT Ab produced by peripheral blood lymphocytes (PBL) in cultures stimulated with TT (6 +/- 2.1 ng/ml vs. 22 +/- 8.4 ng/ml, P less than 0.01); (3) numbers of anti-TT Ab secreting B cells per million cells cultured (6.7 +/- 3.4 vs. 26.6 +/- 7.6, P less than 0.001) and (4) mean ng Ab secreted per anti-TT Ab-secreting B cell (0.6 +/- 0.4 ng vs. 12.7 +/- 7.8 ng, P less than 0.01). This study shows that both decreased numbers of Ag-specific immune B cells and decreased potency on a per cell basis contribute to the impaired immune responses to immunizations in older adults.

Ten to 23% of cells in blood, lymph node and bone marrow from normal dogs formed rosettes with human erythrocytes, and 12-27% formed rosettes with erythrocyte-antibody-complement (EAC) complexes. In contrast, only 3% of thymocytes, and 1% of thoracic duct cells formed rosettes with human erythroyctes, and 0 and 15% respectively formed EAC rosettes. When peripheral blood mononuclear cells were separated by rosette sedimentation into populations depleted of, or enriched for, cells forming rosettes with human erythrocytes (H-RFC), the population depleted of H-RFC responded more vigorously to alloantigens in mixed leukocyte culture (MLC) (P < 0.01) and to the mitogens phytohemagglutinin (PHA) (P = 0.01) and concanavalin A (P = 0.01) than did the population enriched for H-RFC. Passage of peripheral blood mononuclear cells over nylon wool columns produced a nonadherent population depleted of H-RFC, EAC rosette-forming cells and cells binding surface immunoglobulin (SIg), while the adherent population was enriched for each of these markers. In 3 dogs 36%, 44% and 64% of adherent cells that formed rosettes with human erythrocytes also possessed SIg, suggesting that canine B cells form rosettes with human red cells. The nonadherent population showed a more vigorous response to alloantigens in MLC (P < 0.01) and to PHA (P < 0.05) than the adherent population, and also stimulated the growth of autologous erythroid colonies better than the adherent population (P = 0.02). A T cell rich population can thus be obtained from canine peripheral blood, but no specific marker for T cells has been identified. Specifically, the capacity to form rosettes with human red cells is not a marker for the canine T cell.

Twenty-four patients with aplastic anemia or acute leukemia were treated by marrow grafts from HLA-identical donors after conditioning with high doses of cyclophosphamide and/or today body irradiation. They were studied between 4 and 63 mo (median 14.2) after transplantation. Seventeen patients had chronic graft-versus-host disease (C-GVHD) and 7 were healthy. They were studied for defects in their T- and B-cell function using and indirect hemolytic plaque assay for Ig production after 6 days of culture in the presence of pokeweek mitogen. T or B cells from the patients with or without C-GVHD were cocultured with T or B cells from their HLA-identical marrow donors or unrelated normal controls. Intrinsic B-cell defects, lack of helper T-cell activity, and suppressor T-cell activity were more frequently found in patients with C-GVHD than in healthy patients. Fifteen of the 17 patients with C-GVHD showed on or more defects in their T-and B-cell function compared to only 3 of the 7 patients without C-GVHD. None of the healthy controls, including the marrow donors, showed defects in their T- and B-cell functions. These in vitro findings may be helpful in assessing the process of immune reconstitution and the immunologic aberration found after human marrow transplantation.

Peripheral blood lymphocytes from 16 aplastic anemia patients were studied for in vitro biosynthesis of immunoglobulins (Ig), proliferative responses, and cell markers before and after antithymocyte globulin (ATG) treatment in an attempt to identify immune functions that would be useful in predicting responses to ATG therapy. Six of the 16 aplastic anemia patients were complete responders to ATG therapy, two were partial responders, and eight failed to respond to ATG therapy. The proportion of E+, CD4, CD8, and surface Ig-positive cells did not correlate with in vitro lymphocyte functions nor clinical responses before or after ATG therapy. Lymphocyte proliferative responses to phytohemagglutinin, tetanus toxoid, alloantigens, or pokeweed mitogen were generally present before and after ATG therapy. When pokeweed mitogen, herpes simplex type I virus, and tetanus toxoid were used as probes to elicit in vitro Ig production using a hemolytic plaque assay, some patients had 1) B cells that failed to produce Ig, 2) T cells that failed to provide helper activity, and 3) T cells that exhibited excessive suppressor activity in the various antibody production systems. These measures of immune function, however, did not correlate with clinical responses to ATG therapy.