Surface-Enhanced Infrared Spectroscopy and Neutron Reflectivity

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Surface-Enhanced Infrared Spectroscopy and Neutron Reflectivity Studies of Ubiquinone in Hybrid Bilayer Membranes under Potential Control Amanda Quirk,† Michael J. Lardner,† Zin Tun,‡ and Ian J. Burgess*,† †

Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada S7N 5C9 Canadian Neutron Beam Centre, Chalk River Laboratories, Chalk River, ON, Canada K0J 1J0

‡

S Supporting Information *

ABSTRACT: Surface-enhanced infrared adsorption spectroscopy (SEIRAS) and neutron reflectometry (NR) were employed to characterize ubiquinone (UQ) containing hybrid bilayer membranes. The biomimetic membrane was prepared by fusing phospholipid vesicles on a hydrophobic octadecanethiol monolayer self-assembled on a thin gold film. Using SEIRAS, the assembly of the membrane is monitored in situ. The presence of ubiquinone is verified by the characteristic carbonyl peaks from the quinone ester. A well-ordered distal lipid leaflet results from fusion of vesicles with and without the addition of ubiquinone. With applied potential, the hybrid bilayer membrane in the absence of UQ behaves in the same way as previously reported solid supported phospholipid membranes. When ubiquinone is incorporated in the membrane, electric field induced changes in the distal leaflet are suppressed. Changes in the infrared vibrations of the ubiquinone due to applied potential indicate the head groups are located in both polar and nonpolar environments. The spectroscopic data reveal that the isoprenoid unit of the ubiquinone is likely lying in the midplane of the lipid bilayer while the head has some freedom to move within the hydrophobic core. The SEIRAS experiments show redox behavior of UQ incorporated in a model lipid membrane that are otherwise inaccessible with traditional electrochemistry techniques.

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INTRODUCTION

Studies to determine the location of ubiquinone and ubiquinone analogues in a phospholipid membrane both natively and under applied potential have reached varied and conflicting conclusions. A multitude of physical techniques have been used for such studies: differential scanning calorimetry,4−6 fluorescence spectroscopy,4,7−9 X-ray diffraction,10,11 nuclear magnetic resonance,11−15 and neutron diffraction.16 The parameters determining the location of ubiquinone are twofold as a result of its amphiphilic character: the hydrophilic headgroup location (and possible change in location during redox chemistry) and the location and function of the isoprenoid tail. The role of the isoprene domain in ubiquinone mobility, redox reactions, and the location within the

Redox reactions play a vital role in the cells of plants and animals in bioenergetics, signaling, cell metabolism, photosynthesis, and respiration, and thus it is desirable that the structure and conformation of lipids and electron-transport molecules in the membrane matrix be well understood. Determining the location of integral proteins and redox carriers in a biomimetic membrane can contribute to the design of location specific drug delivery. One important example is the role of ubiquinone (UQ or coenzyme Q10) in the electron transport chain.1 Ubiquinone is a lipophilic molecule consisting of a redox-active 2,3dimethoxy-5-methylbenzoquinone ring with a hydrophobic isoprenoid (2-methyl-2-butene) side chain in the 6-position (chemical structure shown in Figure 1). Recent medical and pharmaceutical studies implicate UQ levels in cells as having a profound effect on disease and aging.2,3 © XXXX American Chemical Society

Received: November 19, 2015 Revised: January 8, 2016

A

DOI: 10.1021/acs.langmuir.5b04263 Langmuir XXXX, XXX, XXX−XXX

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describes our efforts to use the isotopic sensitivity of attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) and neutron reflectometry (NR) to investigate the UQ−lipid interaction at different applied potentials. To minimize isotopic scrambling34−36 between the bilayer leaflets, we adopted the hybrid bilayer membrane (HBM) strategy pioneered by Plant37−39 and Knoll40 whereby a lipid monolayer (with or without a small mole fraction of UQ) is transferred by vesicle fusion onto a preformed selfassembled monolayer of an alkanethiol. With access to both deuterated lipid and deuterated thiol we are able to resolve the influence of the UQ and its redox chemistry on the distal and proximal leaflets of the biomimetic bilayer. The lipid−UQ ratios used in this work are slightly higher than most reported biological conditions but comparable to those used in similar biomimetic studies.17,41,42

Figure 1. Chemical structures of (A) ubiquinone and (B) DMPC.

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membrane has not been unequivocally explained. Haines proposed a model whereby any hydrocarbon in the center of lipid bilayers may serve as an inhibitor of proton mobility.17 Additionally, the hydrophobic nature of the lipid bilayer midplane makes it a preferential location for the ubiquinone isoprenoid tail. It has recently been shown that a saturated hydrocarbon analogue of ubiquinone isoprenoid tail inhibits proton leakage across lipid membranes because it lies in the midplane of phospholipid bilayer leaflets.18 Differential scanning calorimetry and fluorescence quenching measurements by Katsikas and Quinn6,9,10 have both shown that ubiquinone (at concentrations up to 10 mol %) lies in between the inner and outer leaflets of a dipalmitoylphosphatidylcholine (DPPC) bilayers. Fluorescence studies by Lenaz et al.19 found the location of ubiquinone oscillates between the two bilayer surfaces but does not extend beyond the glycerol region. A NMR study showed the isoprenoid chain is in a mobile environment, physically separated from the motions and orientational constraints of the lipid acyl chains.11,12 Two additional NMR studies place ubiquinone parallel to the chains of the phospholipids with the quinone head domain in the region of the phospholipid headgroups14 or close to the head groups but distant from the lipid−water interface.13 Neutron diffraction studies by Hauβ et al. indicate the isoprene domain of ubiquinone lies in the midplane bilayer parallel to the phospholipid membrane plane.16 The inconsistent conclusions in previous reports merit additional investigations of the nature of the UQ−lipid interaction. Importantly, the fabrication of a biomimetic lipid layer on a solid support allows the use of new techniques that can distinguish the molecular features of the lipid matrix from those of ubiquinone. Furthermore, if the solid support is conductive, the electric field across the membrane can be precisely controlled, and the UQ can be switched between both biologically relevant forms, quinone and quinol, in an effort to see how its redox state affects the UQ−lipid interaction. Surface sensitive IR spectroscopy20−26 and neutron reflectivity27−31 have been used extensively to study lipid bilayer systems on gold surfaces. Both techniques are highly sensitive to H/D isotopic variation in components of supported lipid bilayers including hydrocarbon backbones32 and solvent distribution in defects and in the headgroup region.33 Careful manipulation of the isotopic composition of the lipid material that comprises the distal and proximal bilayer leaflets can be used to resolve changes in individual contributions induced by a controlled perturbation such as the interfacial potential. The present work

EXPERIMENTAL SECTION

The preparation of a gold film on a polished Si hemisphere for ATRSEIRAS measurements is described elsewhere by Miyake et al.43 with procedural variations detailed by Rosendahl et al.44 The gold film was annealed with a small flamed butane torch for five 15 s intervals, waiting 30 s between each interval. The film was rinsed under a stream of Milli-Q water after annealing and electrochemically cleaned in 0.1 M NaF. The flame annealing was found to improve adhesion of the gold layer to the silicon hemisphere which was empirically shown to result in fewer delamination events over the course of long experiments involving potential and temperature perturbations. SEIRAS measurements were performed on the same gold films before and after annealing. The change in surface enhancement was found to be about 10−20%, which is on the order of differences in enhancement seen from film to film. Surface selection rules on flat metal surfaces are well established for IRAS. Qualitatively, the absorption of radiation by a vibrating molecule is proportional to the square of the electric field strength, and the IR light interacts strongly only with vibrational modes of an adsorbate that have a component of the dipole derivative perpendicular to the surface. The SEIRA-active thin gold films used in the presented experiments are rough and result in random molecular orientations on the macroscopic scale. Osawa et al. show that even on rough surfaces only the vibrations that have dipole changes perpendicular to the metal surface are infrared-active, and enhanced signals result from the adsorbed molecule being excited through the polarization of the metal.45 Alkanethiol self-assembled monolayers were prepared from 2 mM octadecanethiol (hydrogenous, Aldrich, 98%, and perdeuterated, CDN isotopes, 99.1%) solutions in methanol (Fisher, 99.9%) for 17−24 h at room temperature. The thiol SAM was rinsed with methanol and D2O and heated to 40−45 °C in 0.1 mM NaF for 1 h to prepare the system for vesicle fusion. Infrared spectra collected during heating indicate the thiol SAM does not change during this process. Lipid layers were prepared from 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (d54DMPC and DMPC, Avanti Polar Lipids, >99%). Vesicles were prepared by the method described by Barenholz et al.46 Briefly, a 10 mg mL−1 chloroform solution of d54DMPC (or 9:1 mole fraction d54DMPC:UQ) was used as stock solution. Approximately 200 μL of this solution was dried by vortexing in an acid cleaned test tube under a flow of argon. To remove residual solvent, the test tube containing the dried DMPC film was first placed in a vacuum desiccator for at least 2 h, then dipped in liquid nitrogen for 30−60 s, and stored for future use in a vacuum desiccator. The dry lipid vesicles were resuspended in 2 mL of 0.1 M NaF electrolyte prepared in D2O. Specific to the ubiquinone mixtures, the vesicles were prepared by sonicating for 2 h at 50−55 °C, at which point the mixture became translucent. As reported by Ondarroa and Quinn and confirmed here, proper mixing, incorporation, fusion, and retention of ubiquinone into the lipid matrix can only be achieved at temperatures close to 50 °C,47−49 where there is a more favorable free energy of B

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Figure 2. In situ ATR SEIRA spectra during vesicle fusion of d54DMPC:UQ (90:10) vesicles on a hydrogenous SAM forming a hybrid bilayer membrane: (A) carbonyl region, (B) C−D stretch region, and (C) C−H region. Spectra are referenced to octadecanethiol/Au substrate and have been offset by a common amount (0.002) at pinning frequencies of (A) 1550, (B) 2170, and (C) 2800 cm−1. mixing. The solution of vesicles was added to the glass spectroelectrochemical cell containing 2−3 mL of 40−45 °C, 0.1 M NaF using a 1 mL syringe, and a 0.2 μm filter (Filtropur). After 4−5 h, 20 mL of 40 °C, 0.1 NaF was added to the spectroelectrochemical cell, and the counter (coiled gold wire) and reference electrodes (Ag/ AgCl(sat. KCl; D2O)) were introduced. An inert atmosphere of argon and the elevated temperature in the glass cell were maintained for the duration of the experiment. The details of ATR-SEIRAS have been described elsewhere.43,50−53 All SEIRA spectra were measured with a Bruker (Vertex 70) spectrometer equipped with a VeeMAX II ATR accessory (Pike Technologies, Madison, WI). The special resolution was 4 cm−1, and 1024 interferograms were coadded for each spectrum. The sample chamber of the spectrometer was purged throughout the experiment using CO2 and H2O-free air supplied by a Parker Balston FT-IR purge gas generator 75-62 (Parker Hannifin Corporation, Haverhill, MA). For spectra of pure components, a transmission spectrum collected of chloroform solution deposited on a CaF2 window (Figure SI5). For neutron reflectometry experiments, samples were prepared on 12 mm thick, 100 mm diameter single crystal quartz wafers. Measurements were performed at the D3 reflectometer at the Canadian Neutron Beam Centre, Chalk River, Ontario. Experimental details can be found in the Supporting Information. Normal mode analysis of UQ1 was performed using density functional theory (DFT) calculations using the B3LYP functional and the 6-311G++ basis set. Calculations were run on Gaussian 0954 hosted on the Westgrid cluster.

spectra of CH region of octadecanethiol can be found in Figure SI1. The spectra show the characteristic peak shape and positions expected for well-ordered, long-chain, alkanethiol monolayers. Importantly, the methylene asymmetric stretch is below 2918 cm−1, indicating the alkyl chains are wellordered.62,63 The outer leaflet of the HBM was formed through DMPC vesicle fusion with ubiquinone-containing vesicles. It is important to note that elevated temperatures are required for the successful incorporation of ubiquinone into the HBM. Initial SEIRAS experiments performed at room temperature saw successful vesicle fusion on the time scale of 2−3 h; however, at longer times the characteristic infrared absorbances for ubiquinone slowly decrease and then disappear completely. At elevated temperatures (40−45 °C) the ubiquinone remained incorporated in the bilayer for days. The 9:1 molar ratio of lipid to ubiquinone was chosen to create a final bilayer with 5 mol % fraction ubiquinone. Values less than 5 mol % ubiquinone are considered biologically relevant.64 At concentrations higher than 5 mol %, overcrowding in the bilayer is reported, and there is increased segregation into ubiquinone-rich domains.47,48,64 The SEIRA spectra of the UQ-free phospholipid vesicles fusing in situ to the thiol SAM are shown in Figure SI2. The infrared spectra are presented as the change in absorbance referenced to the interface after the addition of electrolyte to the thiol SAM covered Au film. Positive going bands indicate an increase in absorption for a given vibrational mode relative to the reference measurement, due to either a greater number density or an increasing alignment of the transition dipole moment with the electrode’s surface normal. Negative-going peaks (vibrations) indicate a diminishment of the vibration either in density or alignment. Figure 2 shows the evolution of the ATR-SEIRAS response as vesicle fusion proceeds. The spectra were collected every 5 min, and the stable hybrid lipid bilayer was formed after 4−5 h (bold line). Table 1 lists the approximate frequencies of important vibrational modes for the lipid−ubiquinone system. The IR vibrational spectra of the HBM contain three regions of interest. The carbonyl region from 1800−1500 cm−1 contains peaks arising from both lipid choline ester and the quinone moiety of the ubiquinone (Figure 2A). Peaks in the deuterated hydrocarbon region (2250−2000 cm−1) arise from the acyl chains on the lipid (Figure 2B). Finally, the hydrocarbon region from 3000 to 2800 cm−1 arises from the thiol SAM and the ubiquinone isoprenoid chain (Figure 2C). Because of the overlap of vibrational bands in this

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RESULTS AND DISCUSSION Infrared Spectroscopy Studies of Hybrid Bilayer Membrane Formation. Vesicle fusion on self-assembled monolayers (SAM) or Langmuir−Blodgett (LB) monolayers is a highly efficient method for the formation of asymmetric solid supported bilayers. Octadecanethiol (ODT) is a typical choice for hybrid bilayer membrane (HBM) formation due to its ability to form tightly packed and well-ordered monolayers.20,55 Vesicles in aqueous solution have been shown to spontaneously fuse to the hydrophobic surface of alkanethiol SAMs28,37,56,57 as confirmed using surface plasmon resonance,58 cyclic voltammetry,42 and impedance spectroscopy.59 One of the main advantages of using a hybrid bilayer membrane (HBM) is the ability to selectively control the deuteration of the proximal and distal leaflets on the biomimetic membrane. The HBMs studied in this work were formed with a hydrogenous SAM as the inner leaflet and d54DMPC as the outer leaflet (unless otherwise indicated). The self-assembly of alkanethiol monolayers on gold surfaces is a well-understood process, and these layers have been frequently used as a substrate for biological systems.55,60,61 The infrared C

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shown in Figure 3 display characteristic Kiessig fringes, and the results of the fitting analyses are summarized in Table 2

Table 1. Infrared Bands of Interest description

frequency cm−1

general assignment

C−H stretch vas(CH3) vas(CH2) vs(CH3) vs(CH2) CO stretch CO stretch C−O stretch CC stretch C−D stretch vas(CD3) vas(CD2) vs(CD3) vs(CD2)

3000−2800 2960 2924 2872 2854 1740 1650−1679 1490, 1470, 1432, and 1388 1610 2200−2000 2217 2198 2097 2078

lipid: acyl chains methyl asymmetric methylene asymmetric methyl symmetric methylene symmetric lipid: glycerol group ubiquinone: headgroup reduced ubiquinone ubiquinone: headgroup lipid: acyl chains methyl asymmetric methylene asymmetric methyl symmetric methylene symmetric

region, experiments with a perdeuterated octadecanethiol SAM were also performed and are discussed later in the text. The progression in the magnitude of the peaks in Figure 2 stabilizes after 4 h and clearly indicates successful vesicle fusion and the formation of the hybrid phospholipid membrane. The effect of ubiquinone on the phospholipid bilayer membrane, and each of the three IR regions of interest, is discussed in subsequent sections. Neutron Reflectometry Studies of HBM Formation. Neutron reflectometry (NR) was used to verify the formation of hybrid bilayer membranes. The specular reflection of neutrons provides a nondestructive probe of buried interfaces not accessible by other surface sensitive techniques. Hence, NR is particularly well-suited for studying biomimetic films on electrified supports.65 To extract information regarding the interface, the ratio of specularly reflected to incident neutrons of wavelength λ is measured as a function of the momentum transfer vector in the direction normal to the surface. This vector, commonly denoted as Qz, is related to θ as Qz = 4π sin θ/λ, where θ is the grazing angle between the incident beam and the surface of the sample. The standard strategy to analyze NR data is to propose a model of the sample. The model is constructed using known atomic densities and neutron scattering lengths for different isotopes to build a scattering length density (SLD) profile in the direction perpendicular to the surface. The neutron reflectivity curve for the model is calculated using the Parratt algorithm,66 and the model is then subjected to a nonlinear, least-squares fitting routine until the experimental and calculated reflectivity curves agree. The interface in the present system contains several layers including a Ti film used to adhere the gold to the underlying quartz substrate. To minimize the number of fitted parameters, we made NR measurements in the absence of any organic film to fully characterize the metal layers a priori. The results indicated a significant amount of intermixing between the Ti and Au, but repeat measurements made after the electrochemical NR experiments revealed that the metallic film did not change due to further alloying. This enables the thickness and SLD of the metallic layers to be held constant during the analysis of the modified substrate containing the thiol SAM and the HBM. In situ NR measurements were made after the incubation of the hydrogenous thiol SAM (ODT) and following the fusion of h54-DMPC vesicles. In all NR experiments, the electrolyte solvent was a 0.1 M NaF/D2O solution. The reflectivity curves

Figure 3. Reflectivity as a function of perpendicular momentum transfer vector from DMPC/octadecanethiol HBM in D2O/0.1 M NaF (▽), DMPC-UQ(90:10)/octadecanethiol HBM in D2O/0.1 M NaF (□), and octadecanethiol in D2O/0.1 M NaF (○). Inset: Qz range from 0.12 to 0.2 Å.

Table 2. Theoretical and Model Best-Fit Values for Layers in Hybrid Bilayer Membrane theoretical

ODT

layer

SLD

d

quartz Ti Au/Ti Au CH2 region DMPChead

4.186 −1.925 0 to 4a 4.662 −0.36 to 0.1b 3 to 6

∞ 41.0 10.8 76.8 25.3

SLD 4.14 −1.94 1.4 4.5 −0.231 N/A

ODT/DMPC d

SLD

∞ 41.0 10.8 76.8 35.1 9.2

4.14 −1.94 1.4 4.5 −0.05 4.8

a 30−90% Au mixing with Ti. bValue for dense well packed ODT = −0.36; value for lipid tail = +0.1. cd is thickness of film in Å. dSLD is the scattering length density of film in units of 10−6 Å−2.

(corresponding SLD profiles are shown in Figure SI3). Fitting the data for the SAM-only measurement (curve 1, ○, Figure 3) provided an ODT layer thickness of 25 ± 1 Å, which agrees with the value of 23 ± 1 Å reported in previous ellipsometric studies of ODT SAMs in DPPC hybrid bilayer membrane.20 The SLD of the hydrocarbon region is only slightly larger than the theoretical value for a dense SAM, indicating that a wellpacked SAM is formed on the Au surface with few defects. The best fit of the NR data after vesicle fusion revealed that the thickness of the hydrocarbon layer increased to 35.1 Å and required the presence of an additional layer to account for the distal lipid headgroup (curve 2). The thickness of the lipid headgroup is 9.2 Å, which is very comparable to results reported by Meuse et al.20 The total thickness of the HBM was thus 44.3 Å. The overall SLD of the hydrogenous SAM−lipid matrix is slightly higher than the theoretical value because of the incorporation of water.33 The DMPC layer was removed online by rinsing with methanol and electrolyte. The NR data collected thereafter verified that the electrolyte and thiol SAM structure was still D

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Langmuir intact. A hydrogenous DMPC bilayer containing 10 mol % ubiquinone was assembled on the original SAM using vesicle fusion. The resulting reflectivity curve (□) showed only small differences compared to curve 2 (▽) in Figure 3, demonstrating that the presence of UQ does not deleteriously affect vesicle fusion, nor does its presence require an additional layer in the modeling. The SLD of the CH2 region does not change significantly as all hydrocarbons have similar SLDs. The poor SLD contrast between the UQ and the lipid molecules makes it essentially impossible to locate the UQ molecules within the HBM. This lack of contrast can be addressed by using deuterated materials; however, beamtime restrictions prevented these measurements. Reflectivity curves from both the DMPC bilayer and the DMPC-UQ (90:10) bilayer were collected at open circuit potential and at −450 mV. In both cases the resulting curves were identical, indicating the reduction of ubiquinone to ubiquinol does not induce any significant disruption to the lipid structure or water content of the HBM. See Figure SI4 in the Supporting Information for reflectivity curves. Cumulatively, the NR experiments confirm that HBMs are successfully formed and that the UQ molecules are embedded within the resulting bilayer matrix. Effect of Ubiquinone on a Phospholipid Hybrid Membrane Bilayer. Deuterated Hydrocarbon Region: The Phospholipid Acyl Chain. The CD region in Figure 4

supported pure DMPC bilayers.26,32 Figure 4 reveals that a fluid distal leaflet results from vesicle fusion onto the underlying thiol SAM. The presence of UQ in the film has no effect on the vibrational features of the lipid, implying little to no UQ resides in the distal leaflet of the HBM. It is plausible that the lipid in its liquid state would not be effected by the presence of UQ. However, in the case of small molecules such as cholesterol and melitin, even when the supported bilayer is well above its phase transition temperature the incorporation of cholesterol results in noticeable changes in the IR spectra.69,70 Using a phospholipid and ubiquinone system, Quinn and Ondarroa have made similar measurements (with and without UQ in DPPC) and observed the same effect as we note (i.e., nearly identical spectra) and concluded ubiquinone does not readily intercalate between phospholipid molecules. Hydrocarbon Region: The Thiol SAM and Ubquiquione Isoprene Tail. The HBM hydrocarbon stretching region of the HBM has contributions from both the alkanethiol SAM and the UQ isoprenoid chains. Figure 5 shows the spreading of vesicles

Figure 4. Deuterated hydrocarbon region of ATR-SEIRA spectra of HBM composed of d54DMPC:UQ (90:10) () and d54DMPC (- - -) in 0.1 M NaF at ocp. Spectra are referenced to octadecanethiol/Au substrate. Spectra have been offset for clarity.

Figure 5. Hydrocarbon region of ATR-SEIRA spectra of (A) HBM composed of d54DMPC:UQ (90:10) () and d54DMPC (- - -) in 0.1 M NaF at ocp; spectra are referenced to octadecanethiol/Au substrate. (B) IR spectra of hydrocarbon region of pure ubiquinone. Spectra have been offset for clarity.

corresponds to vibrations associated with the deuterated acyl chains of d54DMPC in the outer leaflet of the hybrid lipid bilayer. In assemblies with and without the presence of UQ, the position and magnitude of the characteristic peaks are essentially identical. The appearance of the asymmetric and symmetric methyl (vs(CD3) and vas(CD3)) and methylene (vs(CD2) and vas(CD2)) stretching vibrations is a good indication of successful vesicle fusion. The vas(CD2) and vs(CD2) are located at 2096 ± 0.6 and 2197 ± 0.5 cm−1, respectively, and the vas(CD3) at 2215 ± 1 cm−1 and a very weak vs(CD3) band is found at 2149 cm−1. A Fermi resonance is located at 2073 cm−1. The vas(CD2) and vs(CD2) contain information regarding the conformational order and molecular orientation of the phospholipid chains. Compared to the positions of an all-trans deuterated ODT monolayer, the frequencies present are shifted to slightly higher frequencies.67,68 The shift suggests the presence of gauche conformers in the lipid layer and is consistent with previous studies of solid

on the SAM results in positive bands in the hydrocarbon region irrespective of whether or not it contains any hydrogenous UQ (h-UQ). However, the two spectra in Figure 5 have significant differences in both the relative magnitudes in the peaks their positions. With h-UQ present in the vesicles, the observation of bands in the C−H region is easily attributable to the formation of the outer leaflet with h-UQ present somewhere in the HBM. One might expect that spreading vesicles comprised entirely of d54DMPC (i.e., with no h-UQ) should provide no new signals in the CH region. The dashed line in Figure 5 clearly shows that this is not the case. The small, but appreciable, increase in peak magnitude without ubiquinone can be attributed to subtle reorientations in the SAM acyl chains. Previous studies using surface-enhanced Raman spectroscopy report changes in the alkanethiol layer during bilayer formation.20,71 The increase and peak shift with the addition of lipid vesicles containing ubiquinone is the linear combination of contribuE

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agreement with literature for the distal leaflet of a phospholipid bilayer.22,26,72 Contributions from the UQ headgroup include CO stretching modes between 1640−1670 cm−1 and CC band at 1610 cm−1 and provide independent confirmation of the presence of UQ in the HBM.47,73 The CO stretching vibration band at 1663−1670 cm−1 has previously been found to be sensitive to environment.48,73 Ubiquinone dissolved in nonpolar solvents, such as heptane or cyclopentane, does not have solute−solvent hydrogen bonding, and the carbonyl vibration is consistently located near 1670 cm−1.48 In contrast, when the solvent promotes hydrogen bonding (methanol or water mixtures), the band typically shifts to lower frequencies by ∼4−25 cm−1 depending on the local environment and extent of hydrogen bonding.73 In Figure 6, the CO contribution from the ubiquinone is a broad peak centered at 1650 cm−1 with a full width at half-maximum (fwhm) of 35 cm−1. The broad nature implies that there are contributions from quinone moieties in both lipophilic and polar environments. The small peak at 1610 cm−1 corresponds to the CC stretching mode74,75 of the ubiquinone headgroup. In Figure 6B, a spectrum of pure ubiquinone shows that with random molecular orientation the magnitude of the 1650 and 1610 cm−1 peak are approximately equal.47,73 The relatively weaker signal strength of the 1610 cm−1 in Figure 6A peak may be explained if the quinone headgroups adopt a preferred orientation within the HBM matrix. For a vibrational mode to be IR-active, its transition dipole moment (TDM) must be parallel to the electric field, which is along the direction normal to the Au surface in ATR-SEIRAS. Lamichhane et al. showed that the FTIR spectra of ubiquinones are independent of the number of isoprene units in the chain which means TDMs determined from DFT calculations of relatively small analogues of UQ-10 can provide insight on orientation effects in the current system.76 Calculations were undertaken on UQ1 (one isoprenoid unit) using the B3LYP functional and the 6-311G++ basis set. The atomic displacements of the vibrational modes of UQ1 for the CO mode and the CC coupled to CO mode with labeled transition dipole moments (TDM) are shown in Figure SI 6. While the TDM of the two vibrations are both in the plane of the ring, they are orthogonal to each other. Consequently, the normal component of each TDM will differ depending on the orientation of the ubiquinone headgroup in the HBM. If the headgroup of the ubiquinone lies flat in the hydrophobic core, neither mode is IR-active. If the head is inserted into either lipid leaflet such that the carbonyl is more

tions from changes in the thiol SAM and the addition of UQ isoprenoid chains to the system. The peak positions of vas(CH3) and vs(CH3) are the same for both spectra in Figure 5A at 2960 ± 0.5 and 2872 ± 0.5 cm−1, respectively. In contrast, there is a large shift in peak position (8 and 5 cm−1) and shape for both the vas(CH2) and vs(CH2) peaks when vesicles containing UQ are added to the system. For a thin film of ubiquinone molecules (Figure 5B) the peak positions in the hydrocarbon region are at 2919 and 2850 cm−1, and the spectrum has a similar peak shape as the curve collected for the HBM in Figure 5A. The shift in peak position can be attributed to changes in orientation and confirmation in both the thiol SAM acyl chains and the presence of the isoprenoid chain of the ubiquinone. Carbonyl Region: The Lipid Choline Ester and Ubiquinone Headgroup. The carbonyl region contains contributions from both the choline ester group of the lipid and the carbonyls of the quinone headgroup. Hybrid lipid bilayer systems both with and without ubiquinone show an identical large peak at 1740 cm−1 (Figure 6). This peak position and magnitude is in

Figure 6. Carbonyl region of ATR-SEIRA spectra of (A) HBM composed of d54DMPC:UQ (90:10) () and d54DMPC (- - -) in 0.1 M NaF at ocp; spectra are referenced to octadecanethiol/Au substrate. (B) IR spectra of carbonyl region of pure ubiquinone. Spectra have been offset for clarity.

Figure 7. Infrared spectra of d54DMPC:UQ (90:10) HBM () and d54DMPC (- - -) on hydrogenous SAM held at −450 mV vs Ag/AgCl2 for 60 min: (A) CO region, (B) CD region, (C) CH region. Spectra reference is the HBM at ocp. Spectra have been offset for clarity. F

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Langmuir or less collinear with the surface normal, the CO vibration is active and CC vibration is inactive. The relative intensities of the two signals seen in Figure 6 indicate that a preferred orientation intermediate between these is likely. Cumulatively, the data in Figures 4−6 demonstrate that vesicle fusion results in a hybrid lipid bilayer. Importantly, when ubiquinone is present in the vesicles, it clearly transfers to the HBM but does not perturb the outer leaflet, implying that it penetrates deeper into the hybrid bilayer. Redox of Ubiquinone. After successful formation of a UQcontaining HBM, it is possible to perform electrochemical experiments on the resulting supported bilayer system. To do so, a reference spectrum is first measured at the open circuit potential (ocp). Subsequently, the potential is changed to −450 mV vs Ag/AgCl(sat. KCl) (i.e., more negative of the formal potential of the ubiquinone/ubiquinol redox couple), and new spectra are acquired. The redox of ubiquinone to ubiquinol is a multistep 2H+, 2e− process that is highly dependent on the environment. The redox potential for UQ in water77 has been reported to occur at −0.57 vs Ag/AgCl(sat. KCl) and in a DPPC monolayerbetween −0.3 and −0.5 V vs Ag/AgCl(sat. KCl).41 A potential of −450 mV vs Ag/AgCl(sat. KCl) is sufficiently negative to reduce the ubiquinone without risking the integrity of the gold film. The electroactivity of ubiquinone in the hybrid lipid bilayer drives changes seen in all three regions of interest although the redox of the ubiquinone, and the consequent changes to the HBM are slow.38,78 Figure SI7 shows the change in the carbonyl, deuterated hydrocarbon, and hydrocarbon regions as a function of time. The infrared bands resulting from the reduction of the quinone in Figure 7A show negative absorbance bands at 1666, 1650, and 1610 cm−1. The position and intensities of the CO mode depend on the hydrogen bonding environment and the geometry of the vibration.73 These bands are consistent with the conversion of the quinone to either ubiquinol (2e−, 2H+) or semiubiquinone (1e−) radical. The ∼1666 cm−1 peak is characteristic of the CO stretch of ubiquinone incorporated in a lipid membrane.47,48 Its presence in Figure 7A suggests a significant number of ubiquinone molecules resided in the hydrophobic core of the bilayer but remained accessible for electrochemical reduction. The peak at 1650 cm−1 results from the reduction of quinone in more polar environments, potentially created by local UQ aggregates. Alternatively, as any preparation of a thiol SAM results in pinhole defects in the SAM, the 1650 cm−1 band may result from UQ molecules in a local aqueous environment. It has been suggested by Laval and Majda that the electron transfer needed to fully reduce ubiquinone requires that the UQ leave the lipid layer and diffuse to the nearest pinhole defect in the underlying SAM.79 For completeness, we note that the upward infrared bands arising from ubiquinol are weak and are close to the absorbance cutoff for the Si substrate. Figure SI8 is the 1550−1300 cm−1 region for the d54DMPC:UQ (90:10) HBM on a hydrogenous SAM held at −450 mV vs Ag/AgCl for 60 min. There is one large split peak at 1477 and 1482 cm−1 that can be attributed to a ring CC mode coupled to a C−OH mode.73 When the potential is reversed back to +150 mV, the redox reaction is reversible but slow. Over similar time lengths as the reduction, ∼60 min, the signal of the reformed ubiquinone is almost completely recovered. Similar slow charge transfer through a quinone containing C11 SAM has previously been reported by Ye et al.80

In both HBM assemblies, with applied negative potential there is a peak from the choline ester of the DMPC lipid (solid and dashed lines in Figure 7A). In the d54DMPC HBM (dashed line) the peak is centered at 1740 ± 0.5 cm−1 compared to a much smaller peak at 1743 ± 1 cm−1 with UQ (solid line). Peaks are also present in the deuterated hydrocarbon region (Figure 7B) for the d54DMPC HBM. Without the addition of ubiquinone in the HBM, one would expect the HBM to respond to negative potentials in a similar manner as has been previously reported for solid supported bilayer lipid membranes.32 Garcia-Araez et al. have shown both the outer and inner lipid leaflets adopt a more upright orientation at negative potentials leading to decreases in the absorption of the methyl and methylene vibrations.26 Interestingly, when UQ is incorporated into the HBM, there is no change observed in the deuterated lipid acyl tails at potentials negative enough to induce the redox reaction. This suggests the UQ prevents electric field induced changes to the distal leaflet. One orientation UQ could adopt to restrict proton mobility is with the polyisoprene tail in the center of the bilayer. Previous studies report hydrocarbon chains in cellular membranes81 have an effect on charge transfer, and specifically, it has been observed that the isoprenoid tails of ubiquinone occupy the area in the center of the bilayer.18,42,82 If the tails assume a conformation parallel to the bilayer plane, a monolayer of UQ tails has a footprint16 of 5 nm2 or the area of approximately eight vertically aligned, fully hydrated, DMPC molecules. A 6% mole fraction of UQ in a lipid membrane is sufficient to provide a full surface coverage of the midplane of the lipid bilayer.16 In the presented experiments, 10% mole fraction of UQ introduced through vesicle fusion corresponds to 5% mole fraction in the resulting bilayer and is sufficient to form a complete UQ layer in the bilayer midplane. In the hydrocarbon region for the HBM with and without UQ at −450 mV, both spectra show negative going features (Figure 7C). Without UQ the thiol SAM tilts to a more upright position at −450 mV, similar to the lipid at negative potentials. The applied potential is not sufficiently negative to result in reductive desorption,83 and material leaving the surface as an alternative possibility for the direction of these bands can be ruled out. With UQ incorporated in the HBM there are contributions from both changes in the hydrogenous thiol SAM and from the UQ isoprenoid tail. In both cases there are methyl and methylene contributions and the ubiquinone has additional contributions from the C−H and CC vibration. In an effort to provide more insight into the changes in the hydrocarbon region, the experiment in Figure 7 was repeated with different isotopic composition. Again, d54DMPC:h-UQ (90:10) vesicles were employed, but the thiol SAM was constructed from perdeuterated octadecanethiol (d-ODT). In this way, any spectral features observed in the CH region can be solely attributed to the h-UQ. Figure 8 shows difference spectra for the d54DMPC: h-UQ/d-ODT (- - -) and d54DMPC: h-UQ/h-ODT() HBM at −450 mV referenced to ocp spectra (collected immediately before stepping to negative potentials). The deuterated HBM with hydrogenous UQ (Figure 8 (- - -)) has large downward going bands in the hydrocarbon spectral region. For the isoprenoid analogue squalene, Chun et al. have calculated the contributions in the C−H stretching region (2800−3100 cm−1) from CH3, CH2, and C−H contributions.84 Knowing these assignments, the large loss in spectral intensity can be attributed to a change in orientation in G

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biomimetic membrane, both during assembly and as a function of transmembrane potential. Ubiquinone does not disrupt the outer leaflet of the membrane during assembly of the hybrid bilayer. When a reducing potential is applied, redox-induced changes in the quinone esters are clearly observed. The location of the carbonyl peaks indicates a combination of quinone groups in both polar and nonpolar environments reconciling the conflicting results of previous studies on related systems. The outer leaflet of the hybrid bilayer membrane is completely undisturbed with the reductive potential. The combination of these results leads to the conclusion that most of the isoprenoid ubiquinone tails lie in the midplane space of the bilayer and do not partition into the distal leaflet during assembly of the bilayer or during redox reactions. The quinone head groups occupy different positions in the HBM, specifically the hydrophobic core of the membrane, in small aggregates and in the pinhole defects in the thiol SAM. Assuming this hybrid system is sufficiently biomimetic, the results described herein indicate that upon reduction the quinol preferentially partitions into the intracellular layer of the bilayer membrane which has important implications for proton/electron shuttling across cellular bilayers. Efforts are ongoing in our lab to prepare a more biomimetic system, with the proximal SAM composed of a thiolipid and a distal layer composed of lipids with a range of acyl chain lengths.

Figure 8. Infrared spectra of d54DMPC:UQ (90:10) HBM on perdeuterated SAM (- - -) and hydrogenous SAM (). Reference spectra ocp. Spectra have been offset for clarity.

the vas(CH2) and vs(CH2) at 2927 and 2855 cm−1, respectively, and the v(C−H) at 2959 cm−1 as the tail of ubiquinone moves from a location predominantly parallel to the midplane of the lipid bilayer (and to the gold surface) to a more upright configuration. This change in tilt is a reorientation of the ubiquinone tail toward the metal surface as the tail bends into the proximal SAM. The reorientation would affect the ODT monolayer, but the changes would be very small. In the experiment using perdeuterated ODT (described in Figure 7), weak signals in the C−D stretching region (2000−2200 cm−1) are observed (data not shown). Figure 9 is a cartoon of possible locations of ubiquinone upon formation of the hybrid bilayer membrane and the changes in orientation during electrochemical reduction at negative potentials as determined by this infrared and neutron reflectometry study. Ubiquinone predominantly occupies the midplane space of the as-formed hybrid lipid bilayer (location I in Figure 9A). A number of ubiquinone molecules also likely reside in defects in the SAM and penetrate toward the gold surface (location II in Figure 9A). The SEIRAS experiments indicate that when a negative potential is applied, the ubiquinone reorients such that the tail becomes more upright as it penetrates to variable extents further into the proximal leaflet of the HBM (Figure 9B, I−IV).

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ASSOCIATED CONTENT

* Supporting Information S

This material is available free of charge via the Internet at http://pubs.acs.org/. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04263. Figures SI1−SI8 and Table 1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; phone +1 306 966 4722 (I.J.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Sylvia Fedoruk Canadian Centre for Nuclear Innovation and Innovation Saskatchewan. Research described in this paper was performed at the Canadian Neutron Beam Centre which is supported by the Natural Sciences and Engineering Research Council of Canada and the National Research Council Canada. This research was

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CONCLUSIONS The combination of in situ ATR-SEIRAS and neutron reflectometry has been employed to studied the location of ubiquinone in a hybrid bilayer membrane. The measurements provide new insight into the location of ubiquinone in a

Figure 9. Cartoon of possible locations of ubiquinone in a hybrid bilayer membrane: (A) locations of ubiquinone as formed (at ocp) and (B) locations when a negative potential is applied. H

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(22) Bin, X.; Zawisza, I.; Goddard, J. D.; Lipkowski, J. Electrochemical and PM-IRRAS Studies of the Effect of the Static Electric Field on the Structure of the DMPC Bilayer Supported at a Au(111) Electrode Surface. Langmuir 2005, 21, 330−347. (23) Zawisza, I.; Bin, X.; Lipkowski, J. Potential-Driven Structural Changes in Langmuir-Blodgett DMPC Bilayers Determined by in situ Spectroelectrochemical PM IRRAS. Langmuir 2007, 23, 5180−5194. (24) Lipkowski, J. Building biomimetic membrane at a gold electrode surface. Phys. Chem. Chem. Phys. 2010, 12, 13874−13887. (25) Laredo, T.; Dutcher, J. R.; Lipkowski, J. Electric Field Driven Changes of a Gramicidin Containing Lipid Bilayer Supported on a Au(111) Surface. Langmuir 2011, 27, 10072−10087. (26) Zawisza, I.; Bin, X.; Lipkowski, J. Spectroelectrochemical studies of bilayers of phospholipids in gel and liquid state on Au(111) electrode surface. Bioelectrochemistry 2004, 63, 137−147. (27) Nanda, H. In Proteins in Solution and at Interfaces; Ruso, J. M., Piñeiro, A., Eds.; John Wiley & Sons, Inc.: 2013; Chapter Resolving Membrane-Bound Protein Orientation and Conformation by Neutron Reflectivity, pp 99−111. (28) Krueger, S.; Meuse, C. W.; Majkrzak, C. F.; Dura, J. A.; Berk, N. F.; Tarek, M.; Plant, A. L. Investigation of Hybrid Bilayer Membranes with Neutron Reflectometry: Probing the Interactions of Melittin. Langmuir 2001, 17, 511−521. (29) Wacklin, H. P.; Thomas, R. K. Spontaneous Formation of Asymmetric Lipid Bilayers by Adsorption of Vesicles. Langmuir 2007, 23, 7644−7651. (30) Majkrzak, C.; Berk, N.; Krueger, S.; Dura, J.; Tarek, M.; Tobias, D.; Silin, V.; Meuse, C.; Woodward, J.; Plant, A. First-Principles Determination of Hybrid Bilayer Membrane Structure by PhaseSensitive Neutron Reflectometry. Biophys. J. 2000, 79, 3330−3340. (31) Perez-Salas, U. A.; Faucher, K. M.; Majkrzak, C. F.; Berk, N. F.; Krueger, S.; Chaikof, E. L. Characterization of a Biomimetic Polymeric Lipid Bilayer by Phase Sensitive Neutron Reflectometry. Langmuir 2003, 19, 7688−7694. (32) Garcia-Araez, N.; Brosseau, C. L.; Rodriguez, P.; Lipkowski, J. Layer-by-Layer PMIRRAS Characterization of DMPC Bilayers Deposited on a Au(111) Electrode Surface. Langmuir 2006, 22, 10365−10371. (33) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Electric Field-Driven Transformations of a Supported Model Biological Membrane−An Electrochemical and Neutron Reflectivity Study. Biophys. J. 2004, 86, 1763−1776. (34) Anglin, T. C.; Cooper, M. P.; Li, H.; Chandler, K.; Conboy, J. C. Free Energy and Entropy of Activation for Phospholipid Flip-Flop in Planar Supported Lipid Bilayers. J. Phys. Chem. B 2010, 114, 1903− 1914. (35) Liu, J.; Brown, K. L.; Conboy, J. C. The effect of cholesterol on the intrinsic rate of lipid flip-flop as measured by sum-frequency vibrational spectroscopy. Faraday Discuss. 2013, 161, 45−61. (36) Escoffre, J.-M.; Bellard, E.; Faurie, C.; Sebai, S. C.; Golzio, M.; Teissie, J.; Rols, M.-P. Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 1701−1709. (37) Plant, A. L. Self-assembled phospholipid/alkanethiol biomimetic bilayers on gold. Langmuir 1993, 9, 2764−2767. (38) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Supported phospholipid/alkanethiol biomimetic membranes: insulating properties. Biophys. J. 1994, 67, 1126−1133. (39) Plant, A.; Brighamburke, M.; Petrella, E.; Oshannessy, D. Phospholipid/Alkanethiol Bilayers for Cell-Surface Receptor Studies by Surface Plasmon Resonance. Anal. Biochem. 1995, 226, 342−348. (40) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenhäusser, A. Fusion of Small Unilamellar Lipid Vesicles to Alkanethiol and Thiolipid SelfAssembled Monolayers on Gold. Langmuir 1997, 13, 7085−7091. (41) Hoyo, J.; Guaus, E.; Torrent-Burgués, J.; Sanz, F. Electrochemical behaviour of mixed {LB} films of ubiquinone - {DPPC}. J. Electroanal. Chem. 2012, 669, 6−13.

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DOI: 10.1021/acs.langmuir.5b04263 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.5b04263 Langmuir XXXX, XXX, XXX−XXX