Cholesterol concentration-dependent tBLM admittance change triggered by rVLY.

<p>EIS recorded 30 min after the injection of rVLY. Incubation time 30 min. </p

Modification of Tethered Bilayers by Phospholipid Exchange with Vesicles

Phosphatidylcholine and cholesterol exchange between vesicles and planar tethered bilayer lipid membranes (tBLMs) was demonstrated from electrochemical impedance spectroscopy (EIS), fluorescence microscopy (FM), and neutron reflectometry (NR) data. Cholesterol is incorporated into the tBLMs, as determined by the functional reconstitution of the pore forming toxin α-hemolysin (EIS data), attaining cholesterol concentrations nearly equal to that in the donor vesicles. Using fluorescently labeled lipids and cholesterol, FM indicates that the vesicle–tBLM exchange is homogeneous for the lipids but not for cholesterol. NR data with perdeuterated lipids indicates lipid exchange asymmetry with two lipids exchanged in the outer leaflet for every lipid in the inner leaflet. NR and EIS data further show different exchange rates for cholesterol (<i>t</i>_1/2 < 60 min) and phosphatidylcholine (<i>t</i>_1/2 > 4 h). This work lays the foundation for the preparation of robust, lower defect, more biologically relevant tBLMs by essentially combining the two methods of tBLM formation–rapid solvent exchange and vesicle fusion

Reconstitution of Cholesterol-Dependent Vaginolysin into Tethered Phospholipid Bilayers: Implications for Bioanalysis

<div><p>Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from <i>Gardnerella vaginalis</i> into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity <i>in vitro</i> and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.</p> </div

Admittance change of DOPC/CHOL40% tBLMs by different variants of VLY: rVLY (black bars), rVLY mutants A162E (white bars) and A162V (grey bars).

<p>Exposure time 30 min. </p

rVLY-induced admittance change of DOPC/CHOL 40% tBLMs after 30 min preincubation with different MAbs: neutralizing MAb 9B4 (black bars), moderate neutralizing MAb 10A6 (white bars) and non-neutralizing MAb 21A5 (grey bars).

<p>Exposure of the tBLMs to rVLY time 30 min. rVLY/MAb molar ratios indicated below the abscissa.</p

Impedance Bode plots of DOPC/CHOL40% tBLMs upon exposure to rVLY solutions of different concentrations.

<p>Exposure time 30 min. (A) Impedance magnitude. (B) Impedance phase.</p

Structure and Properties of Tethered Bilayer Lipid Membranes with Unsaturated Anchor Molecules

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [<i>Z</i>-20-(<i>Z</i>-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection–absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative