John C. Conboy

1-Alkyl-3-methylimidazolium bis(perfluoroalkylsulfonyl)imide water-immiscible ionic liquids: The effect of water on electrochemical and physical properties

The electrochemical and physical properties of a class of 1-n-alkyl-3-methylimidazolium (C n mim) bis(perfuoroalkylsulfonyl)imide room-temperature ionic liquids (RTILs) is reported. By varying the chain length on the cation (n = 6, 8, and 10) and the perfluorinated carbon chains of the imide anions, -CF 3 (BMSI) to -CF 2 CF 3 (BETI), the effect on water content, viscosity, conductivity, and potential window has been examined. The water content of the RTILs, both ambient and water-equilibrated, varies linearly with the imidazolium alkyl chain length. The viscosities of the water-equilibrated BMSI- and BETI-based RTILs were accurately calculated using a linear combination of the viscosity of water and the ambient RTILs. The conductivities for this series ranges from 2.9 mS/cm (C 6 mimBMSI) to 0.92 mS/cm (C 10 mimBETI) and were accurately estimated based on the viscosity, density, and molecular weight. The potential windows of the ambient RTILs are 4.3 and 4.9 V for the BMSI and BETI salts, respectively. The window is reduced to 3.9 and 4.3 V upon equilibration with water. The reversible electrochemistry of ferrocene (ΔE P = 70 mV) and decamethylferrocene (ΔE p = 63 mV) has been measured in water-equilibrated imide-based RTILs with as much as 23 mol% H 2 O.

Kinetics and Thermodynamics of Flip-Flop in Binary Phospholipid Membranes Measured by Sum-Frequency Vibrational Spectroscopy

In order to better characterize the dependence of lipid flip-flop rate and thermodynamics on the nature of the lipid headgroup, we have studied the kinetics of flip-flop for single-lipid and mixed-lipid bilayers consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) as a function of both pressure and temperature. The kinetics of flipping were studied by sum-frequency vibrational spectroscopy (SFVS), which does not require exogenous chemical labeling of the lipid species of interest. Additionally, SFVS may be employed to track only a single species (DSPE or DSPC) within a binary mixture by selective deuteration of the matrix lipid to make it spectroscopically inactive. Using this approach, we have found the flip-flop of pure DSPE to be slower than the flip-flop of pure DSPC by nearly 2 orders of magnitude. The thermodynamics of the pure systems were analyzed in order to better understand the physical factors underlying their transmembrane dynamics. Headgroup hydrophobicity and associated solvent effects, as well as lipid packing constraints, appear to play a key role in determining the rate of flip-flop for these two species. For mixtures of DSPE + DSPC, both components exhibited similar rates of flip-flop at a given mole fraction of DSPE. The kinetics and thermodynamics of flip-flop in the mixtures did not vary uniformly with changing composition but were well correlated to changes in the molecular packing as a function of DSPE content in the bilayer.

Lateral Pressure Dependence of the Phospholipid Transmembrane Diffusion Rate in Planar-Supported Lipid Bilayers

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37 degrees C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 x 10(-4) s(-1) to 1.03 x 10(-4) s(-1)) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 +/- 12 A(2)/molecule at 37 degrees C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.

Phospholipid Flip-flop Modulated by Transmembrane Peptides WALP and Melittin

Select transmembrane proteins found in biogenic membranes are known to facilitate rapid bidirectional flip-flop of lipids between the membrane leaflets, while others have no little or no effect. The particular characteristics which determine the extent to which a protein will facilitate flip-flop are still unknown. To determine if the relative polarity of the transmembrane protein segment influences its capacity for facilitation of flip-flop, we have studied lipid flip-flop dynamics for bilayers containing the peptides WALP(23) and melittin. WALP(23) is used as a model hydrophobic peptide, while melittin consists of both hydrophobic and hydrophilic residues. Sum-frequency vibrational spectroscopy (SFVS) was used to characterize the bilayers and determine the kinetics of flip-flop for the lipid component, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), within the mixed bilayers. The kinetic data were utilized to determine the activation thermodynamics for DSPC flip-flop in the presence of the peptides. Melittin was found to significantly reduce the free energy barrier to DSPC flip-flop when incorporated into the bilayer at 1mol.%, while incorporation of WALP(23) at the same concentration led to a more modest reduction of the free energy barrier. The possible mechanisms by which these peptides facilitate flip-flop are analyzed and discussed in terms of the observed activation thermodynamics.

Free Energy and Entropy of Activation for Phospholipid Flip-Flop in Planar Supported Lipid Bilayers

Basic transition state theory is used to describe the activation thermodynamics for phospholipid flip-flop in planar-supported lipid bilayers (PSLBs) prepared by the Langmuir-Blodgett/Langmuir-Schaeffer method. The kinetics of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) flip-flop were determined as a function of temperature and lateral surface pressure using sum-frequency vibrational spectroscopy (SFVS). From the temperature and lateral pressure dependent DSPC flip-flop kinetics, a complete description of the activation thermodynamics for flip-flop in the gel state, including free energy of activation (DeltaG(++)), area of activation (Deltaa(++)), and entropy of activation (DeltaS(++)), was obtained. The free energy barrier for flip-flop of DSPC was determined to be DeltaG(++) = 105 +/- 2 kJ/mol at 40 degrees C at a deposition surface pressure of 30 mN/m. The free energy barrier was found to consist of large opposing entropic and enthalpic contributions. The influence of alkyl chain length on the activation thermodynamics of flip-flop was also investigated. Decreasing the alkyl chain length led to a decrease in DeltaG(++) due primarily to an increase in DeltaS(++). The values obtained here are compared to previous studies investigating flip-flop by vesicle based methods.

Comparison of the energetics of avidin, streptavidin, neutrAvidin, and anti-biotin antibody binding to biotinylated lipid bilayer examined by second-harmonic generation.

A comparison of the binding properties of avidin, streptavidin, neutrAvidin, and antibiotin antibody to a biotinylated lipid bilayer was studied using second-harmonic generation. Protein binding assays were performed on a planar supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 4 mol % biotinylated-cap-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (biotin-cap-DOPE). The equilibrium binding affinities of these biotin-protein interactions were determined, revealing the relative energetic contributions for each protein to the biotinylated lipid ligand. The results show that the binding affinities of avidin, streptavidin, and neutrAvidin for biotin were all strengthened by protein-protein interactions but that the stronger protein-protein interactions observed for streptavidin and neutrAvidin make their binding more energetically favorable. It was also shown that neutrAvidin has the highest degree of nonspecific adsorption to a pure DOPC bilayer, compared to avidin and streptavidin. In addition, the biotin-binding affinity of the antibiotin antibody was found to be of the same order of magnitude as that of avidin, streptavidin, and neutrAvidin. These findings provide important new insights into these biotin-bound protein complexes commonly used in several bioanalytical applications.

The effect of cholesterol on the intrinsic rate of lipid flip–flop as measured by sum-frequency vibrational spectroscopy

Cholesterol is a major constituent of biological membranes in mammalian cells. Experiments have shown that cholesterol influences the physical properties of the plasma membrane, such as lateral diffusion and phase equilibrium. In addition to controlling the 2-dimensional phase behaviour and mobility of lipids in membranes, cholesterol has also been implicated in the transbilayer diffusion of lipids across the bilayer. Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the presence of cholesterol using planar supported lipid bilayer (PSLB) model membranes. Asymmetric PSLBs were prepared using the Langmuir-Blodgett (LB) method by placing a perdeuterated lipid analogue in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of DSPC within the membrane by measuring the decay in the CH3 vs intensity at 2875 cm(-1) with time and as a function of temperature. A complete kinetic analysis of DSPC flip-flop and the effect of cholesterol on the DSPC dynamics are presented. An analysis of the kinetic data in the framework of Eyring theory provides important insight into the transition state enthalpy (deltaH(double dagger)), entropy (deltaS(double dagger)) and free energy (deltaG(double dagger)) for this important biological process. In addition, the transmembrane migration of cholesterol molecules was also explored by SFVS. These combined studies are aimed at providing new insight in to the transbilayer migration of phospholipids and cholesterol in biological membranes and the effects cholesterol plays in membrane dynamics.

Electrostatic induction of lipid asymmetry

The asymmetric arrangement of phospholipids between the two leaflets of the plasma membrane of eukaryotic cells is an integral part of cellular function. ATP-dependent translocases capable of selective lipid transport across the membrane are believed to play a role in this lipid asymmetry, but our understanding of this process is incomplete. Here we show the first direct and quantitative experiments demonstrating the induction of phosphatidylserine asymmetry in a membrane by electrostatic association of poly-l-lysine in an attempt to elucidate the complex factors which govern the establishment and maintenance of lipid compositional asymmetry in the plasma membrane on a fundamental level. The attractive electrostatic interactions between the charged surface-associated polylysine and phosphatidylserine are sufficient to both induce and maintain an asymmetric arrangement of phosphatidylserine in a planar supported membrane, as measured by sum-frequency vibrational spectroscopy. These studies provide a glimpse of the physical and chemical underpinnings of lipid asymmetry in the eukaryotic plasma membrane.

High-throughput screening of drug-lipid membrane interactions via counter-propagating second harmonic generation imaging.

Here we report the use of counter-propagating second harmonic generation (SHG) to image the interactions between the local anesthetic tetracaine and a multicomponent planar supported lipid bilayer array in a label-free manner. The lipid bilayer arrays, prepared using a 3D continuous flow microspotter, allow the effects of lipid phase and cholesterol content on tetracaine binding to be examined simultaneously. SHG images show that tetracaine has a higher binding affinity to liquid-crystalline phase lipids than to solid-gel phase lipids. The presence of 28 mol % cholesterol decreased the binding affinity of tetracaine to bilayers composed of the mixed chain lipid, 1-steroyl-2-oleoyl-sn-glycero-3-phophocholine (SOPC), and the saturated lipids 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC) and 1,2-dipamitoyl-sn-glycero-3-phophocholine (DPPC) while having no effect on diunsaturated 1,2-dioleoyl-sn-glycero-3-phophocholine (DOPC). The maximum surface excess of tetracaine increases with the degree of unsaturation of the phospholipids and decreases with cholesterol in the lipid bilayers. The paper demonstrates that SHG imaging is a sensitive technique that can directly image and quantitatively measure the association of a drug to a multicomponent lipid bilayer array, providing a high-throughput means to assess drug-membrane interactions.

Kinetics and Thermodynamics of Hydrogen Oxidation and Oxygen Reduction in Hydrophobic Room-Temperature Ionic Liquids

In this study 1-dodecyl-3-methylimidazolium (C(12)mim) bis(pentafluoroethylsulfonyl)imide (BETI) and 1-dodecylimidazolium (C(12)im) BETI hydrophobic room-temperature ionic liquids (RTILs) were synthesized and used as proton-conducting electrolytes in a nonhumidified feed gas electrochemical cell. The ionic conductivities of C(12)mimBETI and C(12)imBETI were similar and increased linearly with an increase in temperature from 20 to 130°C. However, when used in the electrochemical system the protic water-equilibrated C(12)imBETI had a larger maximum current and power density compared to the aprotic water-equilibrated C(12)mimBETI. The effect of water content on the reaction rates and thermodynamics of these hydrophobic RTILs was also examined. The efficiency of the C(12)mimBETI increased upon removal of water while that of the C(12)imBETI decreased in efficiency when water was removed. The water structure in these RTILs was examined using attenuated total internal reflection Fourier transform IR spectroscopy and depended on the chemical structure of the cation. These studies give further insight into the possible mechanism of proton transport in these RTIL systems.