Co-reporter:Nelli Teske, Jeremias Sibold, Johannes Schumacher, Nikolas K. Teiwes, Martin Gleisner, Ingo Mey, and Claudia Steinem
Langmuir December 12, 2017 Volume 33(Issue 49) pp:14175-14175
Publication Date(Web):November 17, 2017
DOI:10.1021/acs.langmuir.7b02727
A number of techniques has been developed and analyzed in recent years to generate pore-spanning membranes (PSMs). While quite a number of methods rely on nanoporous substrates, only a few use micrometer-sized pores to be able to individually resolve suspending membranes by means of fluorescence microscopy. To be able to produce PSMs on pores that are micrometer in size, an orthogonal functionalization strategy resulting in a hydrophilic surface is highly desirable. Here, we report on a method to prepare PSMs based on the evaporation of a thin layer of silicon monoxide on top of the porous substrate. PM-IRRAS experiments demonstrate that the final surface is composed of SiOx with 1 < x < 2. The hydrophilic surface turned out to be well suited to spread giant unilamellar vesicles forming PSMs. As the method does not rely on a gold coating as frequently used for orthogonal functionalization, fluorescence micrographs provide information not only from the freestanding membrane areas but also from the supported ones. The observation of the entire PSM area enabled us to observe phase-separation in these membranes on the freestanding and supported parts as well as protein binding and possible lipid reorganization of the membranes induced by binding of the protein Shiga toxin.
Co-reporter:Miriam Schwamborn;Johannes Schumacher;Jeremias Sibold;Nikolas K. Teiwes
Analyst (1876-Present) 2017 vol. 142(Issue 14) pp:2670-2677
Publication Date(Web):2017/07/10
DOI:10.1039/C7AN00215G
Monitoring the proton pumping activity of proteins such as ATPases in reconstituted single proteoliposomes is key to quantify the function of proteins as well as potential proton pump inhibitors. However, most pH-detecting assays available are either not quantitative, require well-adapted reconstitution protocols or are not appropriate for single vesicle studies. Here, we describe the quantitative and time-resolved detection of F-type ATPase-induced pH changes across vesicular membranes doped with the commercially available pH sensitive fluorophore Oregon Green 488 DHPE. This dye is shown to be well suited to monitor acidification of lipid vesicles not only in bulk but also at the single vesicle level. The pKa value of Oregon Green 488 DHPE embedded in a lipid environment was determined to be 6.1 making the fluorophore well suited for a variety of physiologically relevant proton pumps. The TFOF1-ATPase from a thermophilic bacterium was reconstituted into large unilamellar vesicles and the bulk acidification assay clearly reveals the overall activity of the F-type ATPase in the vesicle ensemble with an average pH change of 0.45. However, monitoring the pH changes in individual vesicles attached to a substrate demonstrates that the fraction of vesicles with a significant observable pH change is only about 5%, a number that cannot be gathered from bulk experiments and which is considerably lower than expected.
Co-reporter:U. Rost, C. Steinem and U. Diederichsen
Chemical Science 2016 vol. 7(Issue 9) pp:5900-5907
Publication Date(Web):19 May 2016
DOI:10.1039/C6SC01147K
Transmembrane β-peptide helices and their association in lipid membranes are still widely unexplored. We designed and synthesized transmembrane β-peptides harboring different numbers of D-β3-glutamine residues (hGln) by solid phase peptide synthesis. By means of circular dichroism spectroscopic measurements, the secondary structure of the β-peptides reconstituted into unilamellar vesicles was determined to be similar to a right-handed 314-helix. Fluorescence spectroscopy using D-β3-tryptophan residues strongly suggested a transmembrane orientation. Two or three hGln served as recognition units between the helices to allow helix–helix assembly driven by hydrogen bond formation. The association state of the transmembrane β-peptides as a function of the number of hGln residues was investigated by fluorescence resonance energy transfer (FRET). Therefore, two fluorescence probes (NBD, TAMRA) were covalently attached to the side chains of the transmembrane β-peptide helices. The results clearly demonstrate that only β-peptides with hGln as recognition units assemble into oligomers, presumably trimers. Temperature dependent FRET experiments further show that the strength of the helix–helix association is a function of the number of hGln residues in the helix.
Co-reporter:Oliver Gräb, Maryna Abacilar, Fabian Daus, Armin Geyer, and Claudia Steinem
Langmuir 2016 Volume 32(Issue 39) pp:10144-10152
Publication Date(Web):September 7, 2016
DOI:10.1021/acs.langmuir.6b02575
Long-chain polyamines (LCPAs) are intimately involved in the biomineralization process of diatoms taking place in silica deposition vesicles being acidic compartments surrounded by a lipid bilayer. Here, we addressed the question whether and how LCPAs interact with lipid membranes composed of glycerophospholipids and glyceroglycolipids mimicking the membranes of diatoms and higher plants. Solid supported lipid bilayers and monolayers containing the three major components that are unique in diatoms and higher plants, i.e., monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), were prepared by spreading small unilamellar vesicles. The integrity of the membranes was investigated by fluorescence microscopy and atomic force microscopy showing continuous flat bilayers and monolayers with small protrusions on top of the membrane. The addition of a synthetic polyamine composed of 13 amine groups separated by a propyl spacer (C3N13) results in flat but three-dimensional membrane stacks within minutes. The membrane stacks are connected with the underlying membrane as verified by fluorescence recovery after photobleaching experiments. Membrane stack formation was found to be independent of the lipid composition; i.e., neither glyceroglycolipids nor negatively charged lipids were required. However, the formation process was strongly dependent on the chain length of the polyamine. Whereas short polyamines such as the naturally occurring spermidine, spermine, and the synthetic polyamines C3N4 and C3N5 do not induce stack formation, those containing seven and more amine groups (C3N7, C3N13, and C3N18) do form membrane stacks. The observed stack formation might have implications for the stability and expansion of the silica deposition vesicle during valve and girdle band formation in diatoms.
Co-reporter:Rabindra Nath Das; Y. Pavan Kumar; Ole Mathis Schütte; Claudia Steinem;Jyotirmayee Dash
Journal of the American Chemical Society 2014 Volume 137(Issue 1) pp:34-37
Publication Date(Web):December 16, 2014
DOI:10.1021/ja510470b
A dinucleoside containing guanosine and cytidine at the end groups has been prepared using a modular one-pot azide–alkyne cycloaddition. Single channel analysis showed that this dinucleoside predominantly forms large channels with 2.9 nS conductance for the transport of potassium ions across a phospholipid bilayer. Transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy studies reveal that this dinucleoside can spontaneously associate through Watson–Crick canonical H-bonding and π–π stacking to form stable supramolecular nanostructures. Most importantly, the ion channel activity of this G–C dinucleoside can be inhibited using the nucleobase cytosine.
Co-reporter:Ole M. Schütte, Annika Ries, Alexander Orth, Lukas J. Patalag, Winfried Römer, Claudia Steinem and Daniel B. Werz
Chemical Science 2014 vol. 5(Issue 8) pp:3104-3114
Publication Date(Web):15 May 2014
DOI:10.1039/C4SC01290A
The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb3. In biological membranes, Gb3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb3 complex. To analyse the binding of STxB to different Gb3s, we chemically synthesized saturated, unsaturated, α-hydroxylated Gb3s and a combination thereof, all based on a C24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb3s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescence- and atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb3s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb3s. Large interphase lipid redistribution is observed for α-hydroxylated Gb3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.
Co-reporter:Martin Gleisner, Ingo Mey, Mariam Barbot, Christina Dreker, Michael Meinecke and Claudia Steinem
Soft Matter 2014 vol. 10(Issue 33) pp:6228-6236
Publication Date(Web):27 Jun 2014
DOI:10.1039/C4SM00702F
The generation of a regular array of micrometre-sized pore-spanning membranes that protrude from the underlying surface as a function of osmotic pressure is reported. Giant unilamellar vesicles are spread onto non-functionalized Si/SiO2 substrates containing a highly ordered array of cavities with pore diameters of 850 nm, an interpore distance of 4 μm and a pore depth of 10 μm. The shape of the resulting pore-spanning membranes is controlled by applying an osmotic pressure difference between the bulk solution and the femtoliter-sized cavity underneath each membrane. By applying Young–Laplace's law assuming moderate lateral membrane tensions, the response of the membranes to the osmotic pressure difference can be theoretically well described. Protruded pore-spanning membranes containing the receptor lipid PIP2 specifically bind the ENTH domain of epsin resulting in an enlargement of the protrusions and disappearance as a result of ENTH-domain induced defects in the membranes. These results are discussed in the context of an ENTH-domain induced reduction of lateral membrane tension and formation of defects as a result of helix insertion of the protein in the bilayer.
Co-reporter:Y. Pavan Kumar;Rabindra Nath Das;Sonu Kumar;Ole Mathis Schütte;Dr. Claudia Steinem;Dr. Jyotirmayee Dash
Chemistry - A European Journal 2014 Volume 20( Issue 11) pp:3023-3028
Publication Date(Web):
DOI:10.1002/chem.201304530
Abstract
A “click” ion channel platform has been established by employing a clickable guanosine azide or alkyne with covalent spacers. The resulting guanosine derivatives modulated the traffic of ions across the phospholipid bilayer, exhibiting a variation in conductance spanning three orders of magnitude (pS to nS). Förster resonance energy transfer studies of the dansyl fluorophore with the membrane binding fluorophore Nile red revealed that the dansyl fluorophore is deeply embedded in the phospholipid bilayer. Complementary cytosine can inhibit the conductance of the supramolecular guanosine channels in the phospholipid bilayers.
Co-reporter:Henrik Neubacher, Ingo Mey, Christian Carnarius, Thomas D. Lazzara, and Claudia Steinem
Langmuir 2014 Volume 30(Issue 16) pp:4767-4774
Publication Date(Web):2017-2-22
DOI:10.1021/la500358h
Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 μm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.
Co-reporter:Jan W. Kuhlmann, Ingo P. Mey, and Claudia Steinem
Langmuir 2014 Volume 30(Issue 27) pp:8186-8192
Publication Date(Web):2017-2-22
DOI:10.1021/la5019086
The plasma membrane of animal cells is attached to the cytoskeleton, which significantly contributes to the lateral tension of the membrane. Lateral membrane tension has been shown to be an important physical regulator of cellular processes such as cell motility and morphology as well as exo- and endocytosis. Here, we report on lipid bilayers spanning highly ordered pore arrays, where we can control the lateral membrane tension by chemically varying the surface functionalization of the porous substrate. Surface functionalization was achieved by a gold coating on top of the pore rims of the hexagonal array of pores in silicon nitride substrates with pore radii of 600 nm followed by subsequent incubation with various n-propanolic mixtures of 6-mercapto-1-hexanol (6MH) and O-cholesteryl N-(8′-mercapto-3′,6′-dioxaoctyl)carbamate (CPEO3). Pore-spanning membranes composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine were prepared by spreading giant unilamellar vesicles on these functionalized porous silicon nitride substrates. Different mixtures of 6MH and CPEO3 provided self-assembled monolayers (SAMs) with different compositions as analyzed by contact angle and PM-IRRAS measurements. Site specific force-indentation experiments on the pore-spanning membranes attached to the different SAMs revealed a clear dependence of the amount of CPEO3 in the monolayer on the lateral membrane tension. While bilayers on pure 6MH monolayers show an average lateral membrane tension of 1.4 mN m–1, a mixed monolayer of CPEO3 and 6MH obtained from a solution with 9.1 mol % CPEO3 exhibits a lateral tension of 5.0 mN m–1. From contact angle and PM-IRRAS results, the mole fraction of CPEO3 in solution can be roughly translated into a CPEO3 surface concentration of 40 mol %. Our results clearly demonstrate that the free energy difference between the supported and freestanding part of the membrane depends on the chemical composition of the SAM, which controls the lateral membrane tension.
Co-reporter:Daniel Frese, Siegfried Steltenkamp, Sam Schmitz and Claudia Steinem
RSC Advances 2013 vol. 3(Issue 36) pp:15752-15761
Publication Date(Web):27 Jun 2013
DOI:10.1039/C3RA42723D
Silicon substrates with cavities in the micrometre range were micro-fabricated and appropriately functionalized to allow for the generation of pore-spanning membranes (PSMs) sealing the pore cavities. PSMs were either formed by applying lipids dissolved in organic solvent (painting technique) on a hydrophobically functionalized silicon surface followed by ‘solvent freeze-out’, or solvent-free PSMs were prepared by spreading of giant unilamellar vesicles on hydrophilically functionalized substrates. The geometry of the silicon cavities in conjunction with three dimensional confocal laser scanning microscopy images enabled us to simultaneously monitor the PSMs and a pH-sensitive dye entrapped into the picolitre-sized cavities. The excellent sealing properties of both PSM types allowed an in situ generation of proton gradients across these membranes. In the presence of nigericin, a proton/potassium-antiporter, a preformed potassium ion gradient was transformed into a stable proton gradient across the PSMs, which was visualized by the pH-sensitive dye pyranine entrapped in the silicon cavities in a time resolved manner by means of confocal laser scanning fluorescence microscopy.
Co-reporter:Felix Weiher, Michaela Schatz, Claudia Steinem, and Armin Geyer
Biomacromolecules 2013 Volume 14(Issue 3) pp:
Publication Date(Web):January 31, 2013
DOI:10.1021/bm301737m
Oligomeric Pro-Hyp-Gly- (POG-) peptides, wherein the collagenous triple helix is supported by C-terminal capping, exhibit silica precipitation properties (O, Hyp = (2S,4R)hydroxyproline). As quantified by a molybdate assay, the length of the covalently tethered triple helix (number of POG units) determines the amount of amorphous silica obtained from silicic acid solution. Although lacking charged side chains, the synthetic collagens precipitate large quantities of silicic acid resulting in micrometer-sized spheres of varying surface morphologies as analyzed by scanning electron microscopy. Similar precipitation efficiencies on a fast time scale of less than 10 min were previously described only for biogenic diatom proteins and sponge collagen, respectively, which have a considerably higher structural complexity and limited accessibility. The minicollagens described here provide an unexpected alternative to the widely used precipitation conditions, which generally depend on (poly-)amines in phosphate buffer. Collagen can form intimate connections with inorganic matter. Hence, silica-enclosed collagens have promising perspectives as composite materials.
Co-reporter:Julia A. Braunger, Corinna Kramer, Daniela Morick, and Claudia Steinem
Langmuir 2013 Volume 29(Issue 46) pp:14204-14213
Publication Date(Web):October 21, 2013
DOI:10.1021/la402646k
Phosphoinositides and in particular l-α-phosphatidylinositol-4,5-bisphosphate (PIP2) are key lipids controlling many cellular events and serve as receptors for a large number of intracellular proteins. To quantitatively analyze protein–PIP2 interactions in vitro in a time-resolved manner, planar membranes on solid substrates are highly desirable. Here, we describe an optimized protocol to form PIP2 containing planar solid supported membranes on silicon surfaces by vesicle spreading. Supported lipid bilayers (SLBs) were obtained by spreading POPC/PIP2 (92:8) small unilamellar vesicles onto hydrophilic silicon substrates at a low pH of 4.8. These membranes were capable of binding ezrin, resulting in large protein coverage as concluded from reflectometric interference spectroscopy and fluorescence microscopy. As deduced from fluorescence microscopy, only under low pH conditions, a homogeneously appearing distribution of fluorescently labeled PIP2 molecules in the membrane was achieved. Fluorescence recovery after photobleaching experiments revealed that PIP2 is not mobile in the bottom layer of the SLBs, while PIP2 is fully mobile in the top layer with diffusion coefficients of about 3 μm2/s. This diffusion coefficient was considerably reduced by a factor of about 3 if ezrin has been bound to PIP2 in the membrane.
Co-reporter:Thomas D. Lazzara, Daniela Behn, Torben-Tobias Kliesch, Andreas Janshoff, Claudia Steinem
Journal of Colloid and Interface Science 2012 Volume 366(Issue 1) pp:57-63
Publication Date(Web):15 January 2012
DOI:10.1016/j.jcis.2011.09.067
Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs’ lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin–DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin–DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS–PAGE.Graphical abstractHighlights► Vesicles larger than the pore size of AAO can be used for surface functionalization. ► The vesicle’s lipid composition determines that of the monolayer. ► Receptor lipids are laterally mobile and their density can be readily adjusted. ► Optical transparency of AAO allows label-free in situ analysis of protein binding. ► Functionalized and large surface area allows protein purification from cell lysates.
Co-reporter:Alexer Orth;Dr. Ludger Johannes; Dr. Winfried Römer; Dr. Claudia Steinem
ChemPhysChem 2012 Volume 13( Issue 1) pp:108-114
Publication Date(Web):
DOI:10.1002/cphc.201100644
Abstract
The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. Herein, we show that microscopic phase separation of “raft-like” lipid mixtures in pore-spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb3 were prepared on ordered pore arrays in silicon with pore diameters of 0.8, 1.2 and 2 μm, respectively, by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol derivative rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-ordered- (lo) and liquid-disordered (ld) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb3 (40:35:20:5) membranes. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb3-doped membranes increased the lo-phase considerably and even induced lo-phase domains in non-phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb3 (65:10:20:5).
Co-reporter:Thomas D. Lazzara, Torben-Tobias Kliesch, Andreas Janshoff, and Claudia Steinem
ACS Applied Materials & Interfaces 2011 Volume 3(Issue 4) pp:1068
Publication Date(Web):March 3, 2011
DOI:10.1021/am101212h
Anodic aluminum oxide (AAO) membranes with aligned, cylindrical, nonintersecting pores were selectively functionalized in order to create dual-functionality substrates with different pore-rim and pore-interior surface functionalities, using silane chemistry. We used a two-step process involving an evaporated thin gold film to protect the underlying surface functionality of the pore rims. Subsequent treatment with oxygen plasma of the modified AAO membrane removed the unprotected organic functional groups, i.e., the pore-interior surface. After gold removal, the substrate became optically transparent, and displayed two distinct surface functionalities, one at the pore-rim surface and another at the pore-interior surface. We achieved a selective hydrophobic functionalization with dodecyl-trichlorosilane of either the pore rims or the pore interiors. The deposition of planar lipid membranes on the functionalized areas by addition of small unilamellar vesicles occurred in a predetermined fashion. Small unilamellar vesicles only ruptured upon contact with the hydrophobic substrate regions forming solid supported hybrid bilayers. In addition, pore-rim functionalization with dodecyl-trichlorosilane allowed the formation of pore-spanning hybrid lipid membranes as a result of giant unilamellar vesicle rupture. Confocal laser scanning microscopy was employed to identify the selective spatial localization of the adsorbed fluorescently labeled lipids. The corresponding increase in the AAO refractive index due to lipid adsorption on the hydrophobic regions was monitored by optical waveguide spectroscopy. This simple orthogonal functionalization route is a promising method to control the three-dimensional surface functionality of nanoporous films.Keywords: nanoporous substrates; optical light mode waveguide spectroscopy; orthogonal silanization; pore-spanning lipid membranes; reactive plasma; silane chemistry
Co-reporter:Ines Höfer and Claudia Steinem
Soft Matter 2011 vol. 7(Issue 5) pp:1644-1647
Publication Date(Web):24 Jan 2011
DOI:10.1039/C0SM01429J
Pore-spanning planar membranes on highly ordered porous silicon substrates were shown to be well suited to monitor the calcium ion mediated fusion of large unilamellar vesicles by means of confocal laser scanning fluorescence microscopy and scanning ion conductance microscopy in real-time.
Co-reporter:Thomas D. Lazzara, Christian Carnarius, Marta Kocun, Andreas Janshoff, and Claudia Steinem
ACS Nano 2011 Volume 5(Issue 9) pp:6935
Publication Date(Web):July 28, 2011
DOI:10.1021/nn201266e
Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55–60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5–15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging.Keywords: anodic aluminum oxide; fluorescence microscopy; fluorescence recovery after photobleaching; molecular encapsulation; nanoporous substrates; silane chemistry
Co-reporter:Daniela Behn, Sabine Bosk, Helen Hoffmeister, Andreas Janshoff, Ralph Witzgall, Claudia Steinem
Biophysical Chemistry 2010 150(1–3) pp: 47-53
Publication Date(Web):
DOI:10.1016/j.bpc.2010.02.005
Co-reporter:Anja Bernecker, Joanna Ziomkowska, Svenja Heitmüller, Ralph Wieneke, Armin Geyer and Claudia Steinem
Langmuir 2010 Volume 26(Issue 16) pp:13422-13428
Publication Date(Web):July 19, 2010
DOI:10.1021/la1021627
Ether lipids with alkyl chains of uniform length and varying amine headgroups were synthesized and assembled into bilayer structures in aqueous solution, which served as templates for the formation of silica in two and three dimensions produced under ambient conditions. Dynamic light scattering revealed that unilamellar vesicles of the aminolipids are formed by the extrusion method. The alkylation of the polar amine headgroup was systematically increased from a primary, secondary, and tertiary amine to a quaternary ammonium salt, and the amount of silica was quantified by the β-silicomolybdate method as a function of the headgroup. A lysinol-connected ether lipid harboring two primary amine groups was also investigated. This variation enabled us to compare the influence of the headgroup on the properties of the precipitated silica in detail. By spreading of unilamellar aminolipid vesicles onto planar silicon substrates, two-dimensional planar bilayers can be produced. By means of ellipsometry in conjunction with atomic force microscopy, we were able to demonstrate that very thin silica layers with a thickness of a few nanometers are formed within minutes on the surface of the aminolipid bilayers. All layers are composed of silica nanospheres, and the thickness turned out to be independent of the amine headgroup.
Co-reporter:Andreas Janshoff, Claudia Steinem
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (November 2015) Volume 1853(Issue 11) pp:2977-2983
Publication Date(Web):November 2015
DOI:10.1016/j.bbamcr.2015.05.029
Co-reporter:Rodolfo Briones, Conrad Weichbrodt, Licia Paltrinieri, Ingo Mey, Saskia Villinger, Karin Giller, Adam Lange, Markus Zweckstetter, Christian Griesinger, Stefan Becker, Claudia Steinem, Bert L. de Groot
Biophysical Journal (20 September 2016) Volume 111(Issue 6) pp:
Publication Date(Web):20 September 2016
DOI:10.1016/j.bpj.2016.08.007
The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an “open” channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.
Co-reporter:Ole M. Schütte, Lukas J. Patalag, Lucas M.C. Weber, Annika Ries, Winfried Römer, Daniel B. Werz, Claudia Steinem
Biophysical Journal (16 June 2015) Volume 108(Issue 12) pp:
Publication Date(Web):16 June 2015
DOI:10.1016/j.bpj.2015.05.009
Shiga toxin subunit B (STxB) binding to its cellular receptor Gb3 leads to the formation of protein-lipid clusters and bending of the membrane. A newly developed synthetic route allowed synthesizing the biologically most relevant Gb3-C24:1 2OH species with both, the natural (Gb3-R) as well as the unnatural (Gb3-S) configuration of the 2OH group. The derivatives bind STxB with identical nanomolar affinity, while the propensity to induce membrane tubules in giant unilamellar vesicles is more pronounced for Gb3-S. Fluorescence and atomic force microscopy images of phase-separated supported membranes revealed differences in the lateral organization of the protein on the membrane. Gb3-R favorably induces large and tightly packed protein clusters, while a lower protein density is found on Gb3-S doped membranes.
Co-reporter:Ole M. Schütte, Annika Ries, Alexander Orth, Lukas J. Patalag, Winfried Römer, Claudia Steinem and Daniel B. Werz
Chemical Science (2010-Present) 2014 - vol. 5(Issue 8) pp:NaN3114-3114
Publication Date(Web):2014/05/15
DOI:10.1039/C4SC01290A
The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb3. In biological membranes, Gb3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb3 complex. To analyse the binding of STxB to different Gb3s, we chemically synthesized saturated, unsaturated, α-hydroxylated Gb3s and a combination thereof, all based on a C24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb3s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescence- and atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb3s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb3s. Large interphase lipid redistribution is observed for α-hydroxylated Gb3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.
Co-reporter:U. Rost, C. Steinem and U. Diederichsen
Chemical Science (2010-Present) 2016 - vol. 7(Issue 9) pp:NaN5907-5907
Publication Date(Web):2016/05/19
DOI:10.1039/C6SC01147K
Transmembrane β-peptide helices and their association in lipid membranes are still widely unexplored. We designed and synthesized transmembrane β-peptides harboring different numbers of D-β3-glutamine residues (hGln) by solid phase peptide synthesis. By means of circular dichroism spectroscopic measurements, the secondary structure of the β-peptides reconstituted into unilamellar vesicles was determined to be similar to a right-handed 314-helix. Fluorescence spectroscopy using D-β3-tryptophan residues strongly suggested a transmembrane orientation. Two or three hGln served as recognition units between the helices to allow helix–helix assembly driven by hydrogen bond formation. The association state of the transmembrane β-peptides as a function of the number of hGln residues was investigated by fluorescence resonance energy transfer (FRET). Therefore, two fluorescence probes (NBD, TAMRA) were covalently attached to the side chains of the transmembrane β-peptide helices. The results clearly demonstrate that only β-peptides with hGln as recognition units assemble into oligomers, presumably trimers. Temperature dependent FRET experiments further show that the strength of the helix–helix association is a function of the number of hGln residues in the helix.
