Gintaras Valincius

Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes

Surface-tethered biomimetic bilayer membranes (tethered bilayer lipid membranes (tBLMs)) were formed on gold surfaces from phospholipids and a synthetic 1-thiahexa(ethylene oxide) lipid, WC14. They were characterized using electrochemical impedance spectroscopy, neutron reflection (NR), and Fourier-transform infrared reflection-absorption spectroscopy (FT-IRRAS) to obtain functional and structural information. The authors found that electrically insulating membranes (conductance and capacitance as low as 1 μS cm−2 and 0.6 μF cm−2, respectively) with high surface coverage (>95% completion of the outer leaflet) can be formed from a range of lipids in a simple two-step process that consists of the formation of a self-assembled monolayer (SAM) and bilayer completion by “rapid solvent exchange.” NR provided a molecularly resolved characterization of the interface architecture and, in particular, the constitution of the space between the tBLM and the solid support. In tBLMs based on SAMs of pure WC14, the hexa(ethylene oxide) tether region had low hydration even though FT-IRRAS showed that this region is structurally disordered. However, on mixed SAMs made from the coadsorption of WC14 with a short-chain “backfiller,” ß-mercaptoethanol, the submembrane spaces between the tBLM and the substrates contained up to 60% exchangeable solvent by volume, as judged from NR and contrast variation of the solvent. Complete and stable “sparsely tethered” BLMs (stBLMs) can be readily prepared from SAMs chemisorbed from solutions with low WC14 proportions. Phospholipids with unsaturated or saturated, straight or branched chains all formed qualitatively similar stBLMs.

Structure of functional Staphylococcus aureus α-hemolysin channels in tethered bilayer lipid membranes

We demonstrate a method for simultaneous structure and function determination of integral membrane proteins. Electrical impedance spectroscopy shows that Staphylococcus aureus alpha-hemolysin channels in membranes tethered to gold have the same properties as those formed in free-standing bilayer lipid membranes. Neutron reflectometry provides high-resolution structural information on the interaction between the channel and the disordered membrane, validating predictions based on the channels x-ray crystal structure. The robust nature of the membrane enabled the precise localization of the protein within 1.1 A. The channels extramembranous cap domain affects the lipid headgroup region and the alkyl chains in the outer membrane leaflet and significantly dehydrates the headgroups. The results suggest that this technique could be used to elucidate molecular details of the association of other proteins with membranes and may provide structural information on domain organization and stimuli-responsive reorganization for transmembrane proteins in membrane mimics.

Size-dependent neurotoxicity of β-amyloid oligomers

The link between the size of soluble amyloid beta (Abeta) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Abeta(1-42) resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Abeta(1-42) with a mean particle z-height of 1-2 nm exhibited propensity to bind to phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. A similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Abeta(1-42) oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Abeta(1-42) induced reduction of neuronal cell densities in the CGC cultures.

Structure and properties of tethered bilayer lipid membranes with unsaturated anchor molecules.

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-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.

Electrochemical Impedance Spectroscopy of Tethered Bilayer Membranes

The electrochemical impedance spectra (EIS) of tethered bilayer membranes (tBLMs) were analyzed, and the analytical solution for the spectral response of membranes containing natural or artificially introduced defects was derived. The analysis carried out in this work shows that the EIS features of an individual membrane defect cannot be modeled by conventional electrical elements. The primary reason for this is the complex nature of impedance of the submembrane ionic reservoir separating the phospholipid layer and the solid support. We demonstrate that its EIS response, in the case of radially symmetric defects, is described by the Hankel functions of a complex variable. Therefore, neither the impedance of the submembrane reservoir nor the total impedance of tBLMs can be modeled using the conventional elements of the equivalent electrical circuits of interfaces. There are, however, some limiting cases in which the complexity of the EIS response of the submembrane space reduces. In the high frequency limit, the EIS response of a submembrane space that surrounds the defect transforms into a response of a constant phase element (CPE) with the exponent (α) value of 0.5. The onset of this transformation is, beside other parameters, dependent on the defect size. Large-sized defects push the frequency limit lower, therefore, the EIS spectra exhibiting CPE behavior with α ≈ 0.5, can serve as a diagnostic criterion for the presence of such defects. In the low frequency limit, the response is dependent on the density of the defects, and it transforms into the capacitive impedance if the area occupied by a defect is finite. The higher the defect density, the higher the frequency edge at which the onset of the capacitive behavior is observed. Consequently, the presented analysis provides practical tools to evaluate the defect density in tBLMs, which could be utilized in tBLM-based biosensor applications. Alternatively, if the parameters of the defects, e.g., ion channels, such as the diameter and the conductance are known, the EIS data analysis provides a possibility to estimate other physical parameters of the system, such as thickness of the submembrane reservoir and its conductance. Finally, current analysis demonstrates a possibility to discriminate between the situations, in which the membrane defects are evenly distributed or clustered on the surface of tBLMs. Such sensitivity of EIS could be used for elucidation of the mechanisms of interaction between the proteins and the membranes.

Electrochemical Properties of Nanocrystalline Cadmium Stannate Films

Electrochemical properties of sol-gel nanocrystalline cadmium tin oxide electrodes (CTO) were investigated in 0.2 M potassium chloride buffered at pH 7.4 with phosphate. Films were found to he n-type degenerate semiconductors with charge carrier levels from 10 19 to 10 22 cm 3 depending on the thermal aftertreatment. X-ray diffraction analysis was used to reveal the appearance of the cubic cadmium stannate (Cd 2 SnO 4 ) phase at annealing temperatures above 600°C, and to indicate the extent of this dominant phase above 750°C. The flatband potential (E FB . ) of the film electrodes, as determined from capacitance measurements, was found to be around +0.25 V at pH 7.4. Electrochemical activity toward ten redox processes in the range -0.45 V < E < 0.45 V was investigated, and standard electron transfer rate constants were estimated from ac impedance measurements. The dominant factor in the charge-transfer rate on CTO electrodes is the bulk film charge carrier concentration. It was found that the charge-transfer rates were dependent on the separation of the redox carrier formal potential (E of ) from the CTO flatband potential. The slowest rates (10 5 cm s -1 were found for redox couples where E of E FB . For charge transfer from redox couples where E of is away from E FB , the rates can be several orders of magnitude greater and it is thought that the density of states in the conduction hand is rate limiting.

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 (t1/2 < 60 min) and phosphatidylcholine (t1/2 > 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.

Surface-enhanced Raman spectroscopy for detection of toxic amyloid β oligomers adsorbed on self-assembled monolayers

Surface-enhanced Raman spectroscopy (SERS) was used to detect different spectral features of small (1-2 nm) and large (5-10 nm) synthetic amyloid Aβ-42 oligomers, exhibiting high and no detectable neurotoxicities, respectively. Adsorption of peptides at self-assembled monolayers (SAM) terminated by methyl and pyridinium groups was employed to differentiate toxic and non-toxic oligomers. Three SAMs were analyzed: hydrophobic heptanethiol (HT) and octadecanethiol (ODT) as well as positively charged N-(6-mercapto)hexylpyridinium (MHP) chloride. SERS study revealed twofold adsorption effect, changes in the monolayer structure and appearance of new bands associated with the adsorbed peptides. A band at 1387 cm(-1), observed as a result of the SAM and Aβ-42 interaction, is tentatively assigned to the peptide symmetric stretching vibration of carboxylate groups, and appears to be the most prominent spectral feature distinguishing toxic oligomers from the non-toxic Aβ-42 forms. This band was identified in the spectra of Aβ-42 adsorption on heptanethiol and MHP monolayers, while no clear perturbations were observed in the case of ODT monolayer.

Structure and function of the membrane anchoring self-assembled monolayers.

Structure of the self-assembled monolayers (SAMs) used to anchor phospholipid bilayers to surfaces affects the functional properties of the tethered bilayer membranes (tBLMs). SAMs of the same surface composition differing in the lateral distribution of the anchor molecule give rise to tBLMs of profoundly different defectiveness with residual conductance spanning 3 orders of magnitude. SAMs composed of anchors containing saturated alkyl chains, upon exposure to water (72 h), reconstruct to tightly packed clusters as deduced from reflection absorption infrared spectroscopy data and directly visualized by atomic force microscopy. The rearrangement into clusters results in an inability to establish highly insulating tBLMs on the same anchor layer. Unexpectedly, we also found that nanometer scale smooth gold film surfaces, populated predominantly with (111) facets, exhibit poor performance from the standpoint of the defectiveness of the anchored phospholipid bilayers, while corrugated (110) dominant surfaces produced SAMs with superior tethering quality. Although the detailed mechanism of cluster formation remains to be clarified, it appears that smooth surfaces favor lateral translocation of the molecular anchors, resulting in changes in functional properties of the SAMs. This work unequivocally establishes that conditions that favor cluster formation of the anchoring molecules in tBLM formation must be identified and avoided for the functional use of tBLMs in biomedical and diagnostic applications.

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

Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from Gardnerella vaginalis 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 in vitro 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.