Effect of Lipid Composition on the Inhibition Mechanism of Amiloride on Alamethicin Ion Channels in Supported Phospholipid Bilayers.

The inhibition effect of amiloride on alamethicin ion channels was studied in a model zwitterionic floating bilayer lipid membrane (fBLM). The EIS studies indicated that amiloride prevents the transport of ions through the alamethicin channels leading to an overall increase in membrane resistance. The PM-IRRAS data demonstrated that amiloride has no influence on the secondary structure of alamethicin but restricts the insertion of the peptides into the bilayer and blocks ion transport through preformed alamethicin channels. The effect of amiloride on ion channel formation in the floating bilayer formed by a zwitterionic lipid was compared to those of previous studies involving negatively charged fBLMs and tethered zwitterionic lipid bilayers. The findings from these studies show that the effects of amiloride on ion channel formation strongly depend on the mobility and charge of the membrane lipids.

Binding Affinity of Concanavalin A to Native and Acid-Hydrolyzed Phytoglycogen Nanoparticles.

Phytoglycogen (PG) is a polysaccharide produced in the kernels of sweet corn as soft, highly branched, compact nanoparticles. Its tree-like or dendritic architecture, combined with a high-safety profile, makes PG nanoparticles attractive for use in biological applications, many of which rely on the association or binding of small biomolecules. We have developed a methodology to functionalize surface plasmon resonance (SPR) sensor surfaces with PG nanoparticles, and we demonstrate the utility of the PG-functionalized SPR sensor by measuring the binding affinity of the tetrameric concanavalin A (ConA) protein to both native PG nanoparticles and smaller, softer acid-hydrolyzed PG nanoparticles. We measure comparable values of the equilibrium association constant K for native and acid-hydrolyzed PG, with a slightly smaller value for the acid-hydrolyzed particles that we attribute to unfavorable lateral interactions between the tetrameric subunits of ConA due to the increase in surface curvature of the smaller acid-hydrolyzed PG particles. We also use infrared reflection-absorption spectroscopy (IRRAS) to show that ConA maintains a large fraction of its native conformation, and thus its bioactivity, upon binding to PG, representing an important step toward the realization of PG as a novel bioactive delivery vehicle.

Ion-Pairing Mechanism for the Valinomycin-Mediated Transport of Potassium Ions across Phospholipid Bilayers.

The role of the anion on the ionophore properties of valinomycin was studied in a model floating bilayer lipid membrane (fBLM) using supporting electrolytes containing K+ with four different counter anion species (ClO4-, H2PO4-, Cl-, and F-). The electrochemical impedance spectra indicate that the membrane resistance of the bilayer decreases with the decrease of Gibbs free energy of anion solvation. The IR spectra demonstrate that valinomycin does not readily bind to K+ in the KH2PO4, KCl, and KF electrolyte solutions, but in the presence of KClO4, valinomycin readily binds to K+, forming a valinomycin-K+ complex. The results in the present paper reveal the role of the counter anion on the transport of cations by valinomycin across the lipid bilayer. The valinomycin-cation complex creates an ion pair with the anion, and this ion pair can enter the hydrophobic region of the bilayer transporting the cation across the membrane. Anions with low solvation energies facilitate the formation of the ion pair improving the ion conductivity of valinomycin-incorporated bilayers. This paper sheds new light on the transport mechanism of valinomycin ionophores and provides new information about the bioactivity of this molecule.

Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View.

The effects of amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane.

Electric-Field-Driven Molecular Recognition Reactions of Guanine with 1,2-Dipalmitoyl-sn-glycero-3-cytidine Monolayers Deposited on Gold Electrodes.

Monolayers of 1,2-dipalmitoyl-sn-glycero-3-cytidine were incubated with guanine in a 0.1 M NaF electrolyte at the surface of a Langmuir trough and transferred to gold (111) electrodes using the Langmuir-Schaefer technique. Chronocoulometry and photon polarization modulation infrared reflection absorption spectroscopy were employed to investigate the influence of the static electric field on the orientation and conformation of the cytidine nucleolipid molecules on the metal surface in the presence of guanine and to monitor the molecular recognition of guanine with the cytosine moiety. When the monolayer is exposed to guanine solutions, the cytosine moiety binds to the guanine residue in either a Watson-Crick complex at positively charged electrode surfaces or a noncomplexed state at negative surface charges. The positive electrostatic field causes the cytosine moiety and the cytosine-guanine complex to adopt a nearly parallel orientation with respect to the plane of the monolayer with a measured tilt angle of ∼10°. The parallel orientation is stabilized by the interactions between the permanent dipole of the cytosine moiety or the Watson-Crick complex and the static electric field. At negative charge densities, the tilt of the cytosine moiety increases by ∼15-20°, destabilizing the complex. Our results demonstrate that the static electric field has an influence on the molecular recognition reactions between nucleoside base pairs at the metal-solution interface and can be controlled by altering the surface charge at the metal.

How Valinomycin Ionophores Enter and Transport K+ across Model Lipid Bilayer Membranes.

Valinomycin, a cyclic peptide, was incorporated into a biomimetic lipid membrane tethered to the surface of a gold (111) electrode. Electrochemical impedance spectroscopy was used to study the ionophore properties of the peptide, and polarization modulation infrared reflection absorption spectroscopy was employed to determine the conformation and orientation of valinomycin in the membrane. The combination of these two techniques provided unique information about the ionophore mechanism where valinomycin transports ions across the membrane by creating a complex with potassium ions and forming an ion pair with a counter anion. The ion pair resides within the hydrophobic fragment of the membrane and adopts a small angle of ∼22° with respect to the surface normal. This novel study provides new insights explaining the valinomycin ion transport mechanism in model biological membranes.

Role of Transmembrane Potential and Defects on the Permeabilization of Lipid Bilayers by Alamethicin, an Ion-Channel-Forming Peptide.

The insertion and ion-conducting channel properties of alamethicin reconstituted into a 1,2-di- O-phytanyl- sn-glycero-3-phosphocholine bilayer floating on the surface of a gold (111) electrode modified with a 1-thio-ß-d-glucose (ß-Tg) self-assembled monolayer were investigated using a combination of electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The hydrophilic ß-Tg monolayer separated the bilayer from the gold substrate and created a water-rich spacer region, which better represents natural cell membranes. The EIS measurements acquired information about the membrane resistivity (a measure of membrane porosity), and the PM-IRRAS experiments provided insight into the conformation and orientation of the membrane constituents as a function of the transmembrane potential. The results showed that the presence of alamethicin had a small effect on the conformation and orientation of phospholipid molecules within the bilayer for all studied potentials. In contrast, the alamethicin peptides assumed a surface state, where the helical axes adopted a large tilt angle with respect to the surface normal, at small transmembrane potentials, and inserted into the bilayer at sufficiently negative transmembrane potentials forming pores, which behaved as barrel-stave ion channels for ionic transport across the membrane. The results indicated that insertion of alamethincin peptides into the bilayer was driven by the dipole-field interactions and that the transitions between the inserted and surface states were electrochemically reversible. Additionally, the EIS measurements performed on phospholipid bilayers without alamethicin also showed that the application of negative transmembrane potentials introduces defects into the bilayer. The membrane resistances measured in both the absence and presence of alamethicin show similar dependencies on the electrode potential, suggesting that the insertion of the peptide may also be assisted by the electroporation of the membrane. The findings in this study provide new insights into the mechanism of alamethicin insertion into phospholipid bilayers.

Pore Forming Properties of Alamethicin in Negatively Charged Floating Bilayer Lipid Membranes Supported on Gold Electrodes.

Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-ß-d-glucose (ß-Tg). The EIS data showed that the presence of alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.

Quantitative Subtractively Normalized Interfacial Fourier Transform Infrared Reflection Spectroscopy Study of the Adsorption of Adenine on Au(111) Electrodes.

Quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was used to determine the molecular orientation and identify the metal-molecular interactions responsible for the adsorption of adenine from the bulk electrolyte solution onto the surface of the Au(111) electrode. The recorded p-polarized IR spectra of the adsorbed species were subtracted from the collected s-polarized IR spectra to remove the IR contributions of the vibrational bands of the desorbed molecules that are located within the thin layer cavity of the spectroelectrochemical cell. The intense IR band around 1640 cm(-1), which is assigned to the pyrimidine ring stretching vibrations of the C5-C6 and C6-N10 bonds, and the IR band at 1380 cm(-1), which results from a combination of the ring stretching vibration of the C5-C7 bond and the in-plane CH bending vibration, were selected for the quantitative analysis measurements. The transition dipoles of these bands were evaluated by DFT calculations. Their orientations differed by 85 ± 5°. The tilt angles of adsorbed adenine molecules were calculated from the intensity of these two vibrations at different potentials. The results indicate that the molecular plane is tilted at an angle of 40° with respect to the surface normal of the electrode and rotates by 16° around its normal axis with increasing electrode potential. This orientation results from the chemical interaction between the N10 and gold atoms coupled with the π-π parallel stacking interactions between the adjacent adsorbed molecules. Furthermore, the changes in the molecular plane rotation with the electric field suggests that the N1 atom of adenine must also participate in the interaction between the molecule and metal.

PM-IRRAS Studies of DMPC Bilayers Supported on Au(111) Electrodes Modified with Hydrophilic Monolayers of Thioglucose.

A phospholipid bilayer composed of 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (d54-DMPC) was deposited onto the Au(111) electrode modified with a self-assembled monolayer of 1-thio-ß-d-glucose (ß-Tg) via the Langmuir-Blodgett and Langmuir-Schaefer (LB-LS) techniques. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane on a hydrophilic surface modified gold electrode. The results of the spectroscopic measurements showed that the tilt angle of acyl chains obtained for deuterated DMPC bilayers supported on the ß-Tg-modified gold is significantly lower than that reported previously for DMPC bilayers deposited directly on Au(111) electrodes. Moreover, tilt angles of ∼18° were obtained for d54-DMPC bilayers on ß-Tg self-assembled monolayers (SAMs) at positive potentials, which are similar to the values calculated for h-DMPC deposited on bare gold in the desorbed state and to those observed for a stack of hydrated DMPC bilayers. This data confirms that the ß-thioglucose SAM promotes the formation of a water cushion that separates the phospholipid bilayer from the metal surface. As a result, the DMPC polar heads are not in direct contact with the electrode and can adopt a zigzag configuration, which strengthens the chain-chain interactions and allows for an overall decrease in the tilt of the acyl chains. These novel supported model membranes may be especially useful in studies pertaining to the incorporation of peptides and proteins into phospholipid bilayers.

Quantitative SHINERS analysis of temporal changes in the passive layer at a gold electrode surface in a thiosulfate solution.

Shell-isolated gold nanoparticles (SHINs) were employed to record shell-isolated nanoparticle-enhanced Raman spectra (SHINERS) of a passive layer formed at a gold surface during gold leaching from thiosulfate solutions. The (3-aminopropyl)triethoxysilane (APTES) and a sodium silicate solution were used to coat gold nanoparticles with a protective silica layer. This protective silica layer prevented interactions between the thiosulfate electrolyte and the gold core of the SHINs when the SHINs-modified gold electrode was immersed into the thiosulfate lixiviant. The SHINERS spectra of the passive layer, formed from thiosulfate decomposition, contained bands indicative of hydrolyzed APTES. We have demonstrated how to exploit the presence of these APTES bands as an internal standard to compensate for fluctuations of the surface enhancement of the electric field of the photon. We have also developed a procedure that allows for removal of the interfering APTES bands from the SHINERS spectra. These methodological advancements have enabled us to identify the species forming the passive layer and to determine that the formation of elemental sulfur, cyclo-S8, and polymeric sulfur chains is responsible for inhibition of gold dissolution in oxygen rich thiosulfate solutions.

SEIRAS Studies of Water Structure in a Sodium Dodecyl Sulfate Film Adsorbed at a Gold Electrode Surface.

Surface-enhanced infrared reflection-absorption spectroscopy (SEIRAS) was used to investigate the structure of water that is incorporated within a film of sodium dodecyl sulfate (SDS) adsorbed at a thin gold nanoparticle film deposited onto a silicon substrate. Previous studies on a Au(111) electrode surface showed that SDS molecules form long-range ordered hemicylindrical hemimicelles (phase I) for potentials -0.2 ≤ E ≤ 0.45 V vs Ag/AgCl and a disordered bilayer (phase II) for potentials E ≥ 0.5 V vs Ag/AgCl. The SEIRA spectra demonstrated that the hemimicellar film is water-rich and contains both a network of hydrogen-bonded water and a disturbed network of hydrogen bonds consisting of monomeric and dimeric water in the hydrophobic region of the film. No network water was observed in phase II of the film. However, SEIRAS data showed that sulfate groups in the disordered bilayer are hydrated. The SEIRAS spectra of the film of SDS were compared to the previously measured spectra obtained using subtractively normalized interfacial Fourier transform IR spectroscopy (SNIFTIRS). The complementarity of the spectroscopic information obtained by these two techniques was demonstrated.

Spectroscopic and permeation studies of phospholipid bilayers supported by a soft hydrogel scaffold.

Polarized attenuated total reflection infrared (ATR-IR) spectroscopy, fluorescence microscopy, and fluorescence spectroscopy were used to characterize a lipid coating composed of 70 mol % 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 30 mol % cholesterol, supported on a spherical hydrogel scaffold. The fluorescence microscopy images show an association between the lipid coating and the hydrogel scaffold. Fluorescence permeability measurements revealed that the phospholipid coating acts as a permeability barrier, exhibiting characteristics of a lamellar bilayer coating structure. Variable evanescent wave penetration depth ATR-IR spectroscopy studies validated the determination of quantitative molecular orientation information for a lipid coating supported on a spherical scaffold. These polarized ATR-IR studies measured an average DMPC acyl chain tilt angle of ∼21-25°, with respect to the surface normal.

Electrochemical and PM-IRRAS characterization of cholera toxin binding at a model biological membrane.

A mixed phospholipid-cholestrol bilayer, with cholera toxin B (CTB) units attached to the monosialotetrahexosylganglioside (GM1) binding sites in the distal leaflet, was deposited on a Au(111) electrode surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane upon binding CTB and the application of the electrode potential. The data presented in this article show that binding cholera toxin to the membrane leads to an overall increase in the tilt angle of the fatty acid chains; however, the conformation of the bilayer remains relatively constant as indicated by the small decrease in the total number of gauche conformers of acyl tails. In addition, the bound toxin caused a significant decrease in the hydration of the ester group contained within the lipid bilayer. Furthermore, changes in the applied potential had a minimal effect on the overall structure of the membrane. In contrast, our results showed significant voltage-dependent changes in the average orientation of the protein α-helices that may correspond to the voltage-gated opening and closing of the central pore that resides within the B subunit of cholera toxin.

Application of PM-IRRAS to study thin films on industrial and environmental samples.

Polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS) was employed to analyze two unique samples: (1) an industrially prepared alkoxysilane-pretreated aluminum alloy (AA6111) in the absence and presence of a ~600-nm-thick lubricant coating and (2) a chemical warfare agent simulant, triethyl phosphate (TEP), on glass. For the pretreated aluminum samples, PM-IRRAS spectra were analyzed for three distinct regions; the SiO stretching vibration around 1120 cm(-1), the NH(2) bending mode at ~1600 cm(-1) and the CH stretching region around 2900 cm(-1). Our results showed that increasing the curing temperature (from 55 to 100 °C) improved the overall extent of cross-linking within the siloxane network. In addition, the spectra of lubricant (top coating) and the underlying siloxane layer for the aluminum samples with lubricant were collected for the same sample. Our results show that the nature of the siloxane film remains intact and unaltered after deposition of the lubricant top-coat. For detection of TEP on glass, the band at 1268 cm(-1), corresponding to the P═O vibration, was monitored. A droplet of TEP solution in dichloromethane was deposited on glass. After solvent evaporation had occurred, the intensity of the P═O vibration band was used to construct calibration curves to determine the experimental limit of detection, which was found to be ~200 µg for TEP on glass.

Infrared studies of the potential controlled adsorption of sodium dodecyl sulfate at the Au(111) electrode surface.

Quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was used to determine the conformation and orientation of sodium dodecyl sulfate (SDS) molecules adsorbed at the single crystal Au(111) surface. The SDS molecules form a hemimicellar/hemicylindrical (phase I) structure for the range of potentials between -200 ≤ E < 450 mV and condensed (phase II) film for electrode potentials ≥500 mV vs Ag/AgCl. The SNIFTIRS measurements indicate that the alkyl chains within the two adsorbed states of SDS film are in the liquid-crystalline state rather than the gel state. However, the sulfate headgroup is in an oriented state in phase I and is disordered in phase II. The newly acquired SNIFTIR spectroscopy measurements were coupled with previous electrochemical, atomic force microscopy, and neutron reflectivity data to improve the current existing models of the SDS film adsorbed on the Au(111) surface. The IR data support the existence of a hemicylindrical film for SDS molecules adsorbed at the Au(111) surface in phase I and suggest that the structure of the condensed film in phase II can be more accurately modeled by a disordered bilayer.

Molecular recognition between guanine and cytosine-functionalized nucleolipid hybrid bilayers supported on gold (111) electrodes.

A hybrid bilayer lipid membrane (hBLM), constructed with a 1-hexadecanethiol self-assembled interior leaflet and a 1,2-dipalmitoyl-sn-glycero-3-cytidine nucleolipid exterior leaflet, was deposited at the surface of a gold (111) electrode. This system was used to investigate the molecular recognition reaction between the cytosine moieties of the lipid head group with guanine molecules in the bulk electrolyte solution. Electrochemical measurements and photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) were employed to characterize the system and determine the extent of the molecular recognition reaction. The capacitance of the hBLM-covered gold electrode was very low (~1 µF cm-2), therefore the charge density at the gold surface was small. Changing the electrode potential had a minimal effect on the complexation between the cytosine moieties and guanine molecules due to small changes in the static electric field across the membrane. This behavior favored the formation of the guanine-cytosine complex.