Determination of a refractive index and an extinction coefficient of standard production of CVD-graphene

The refractive index and extinction coefficient of chemical vapour deposition grown graphene are determined by ellipsometry analysis. Graphene films were grown on copper substrates and transferred as both monolayers and bilayers onto SiO2/Si substrates by using standard manufacturing procedures. The chemical nature and thickness of residual debris formed after the transfer process were elucidated using photoelectron spectroscopy. The real layered structure so deduced has been used instead of the nominal one as the input in the ellipsometry analysis of monolayer and bilayer graphene, transferred onto both native and thermal silicon oxide. The effect of these contamination layers on the optical properties of the stacked structure is noticeable both in the visible and the ultraviolet spectral regions, thus masking the graphene optical response. Finally, the use of heat treatment under a nitrogen atmosphere of the graphene-based stacked structures, as a method to reduce the water content of the sample, and its effect on the optical response of both graphene and the residual debris layer are presented. The Lorentz-Drude model proposed for the optical response of graphene fits fairly well the experimental ellipsometric data for all the analysed graphene-based stacked structures.

Translocation of inserted foreign epitopes by a channelforming protein

Certain bacterial protein toxins are able to insert themselves into, and at least partially across, lipid bilayer membranes in the absence of any auxiliary proteins, by using unknown mechanisms to overcome the high energy barrier presented by the hydrophobic bilayer core. We have previously shown that one such toxin, colicin Ia, translocates a large, hydrophilic part of itself completely across a lipid bilayer in conjunction with the formation of an ion-conducting channel. To address the question of whether the colicin can translocate any arbitrary amino acid sequence, we have altered the translocated segment by inserting, singly, two different foreign epitopes. Colicins containing either epitope retain significant bactericidal activity and form channels of normal conductance in planar bilayers. Furthermore, antibodies added on the side of the bilayer opposite that to which the colicin was added interact specifically with the corresponding epitopes, producing an inhibition of channel closing. Thus, the inserted epitopes are translocated along with the rest of the segment, suggesting that a surprisingly small part of colicin Ia, located elsewhere in the molecule, acts as a nonspecific protein translocator.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted Mr 2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.

Rapid and efficient fusion of phospholipid vesicles by the α-helical core of a SNARE complex in the absence of an N-terminal regulatory domain

A protease-resistant core domain of the neuronal SNARE complex consists of an α-helical bundle similar to the proposed fusogenic core of viral fusion proteins [Skehel, J. J. & Wiley, D. C. (1998) Cell 95, 871–874]. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores (≈10 min) is compatible with the kinetics of fusion in many cell types.

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl− secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl− secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl− secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl− secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl− secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl− selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl−-selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Role of lipid polymorphism in G protein-membrane interactions: Nonlamellar-prone phospholipids and peripheral protein binding to membranes

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein α-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein β-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.

The conducting form of gramicidin A is a right-handed double-stranded double helix

The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDHℛ) reported here agree with 15N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs+ and K+ salts may be in error and that our evidence points to the DSDHℛ as the major conformer responsible for ion transport in membranes.

Chiral molecular self-assembly of phospholipid tubules: A circular dichroism study

We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure.

Network formation of lipid membranes: Triggering structural transitions by chain melting

Phospholipids when dispersed in excess water generally form vesicular membrane structures. Cryo-transmission and freeze-fracture electron microscopy are combined here with calorimetry and viscometry to demonstrate the reversible conversion of phosphatidylglycerol aqueous vesicle suspensions to a three-dimensional structure that consists of extended bilayer networks. Thermodynamic analysis indicates that the structural transitions arise from two effects: (i) the enhanced membrane elasticity accompanying the lipid state fluctuations on chain melting and (ii) solvent-associated interactions (including electrostatics) that favor a change in membrane curvature. The material properties of the hydrogels and their reversible formation offer the possibility of future applications, for example in drug delivery, the design of structural switches, or for understanding vesicle fusion or fission processes.

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu2–51 (residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu28–81 (residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu28–81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Channel formation by antiapoptotic protein Bcl-2

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic α-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Δh5, 6) mutant did not. The most frequent conductance observed (18 ± 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ± 2 pS and 90 ± 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Inhibition of Secretion by 1,3-Cyclohexanebis(methylamine), a Dibasic Compound That Interferes with Coatomer Function

We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the ε-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1,3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane.