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Determination of the Rate Limiting Step in Fatty Acid Transport Victoria Cheng, Dylan R. Kimball, and John C. Conboy J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b05162 • Publication Date (Web): 23 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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The Journal of Physical Chemistry

Determination of the Rate Limiting Step in Fatty Acid Transport Victoria Cheng, Dylan R. Kimball, and Dr. John C. Conboy* University of Utah, Department of Chemistry, 315 South 1400 East, RM 2020, Salt Lake City, UT 84112 *[email protected]

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2 Abstract

The generally accepted model of free fatty acid (FA) transport through cellular membranes occurs in three steps, adsorption of the FA onto the membrane, translocation across the membrane (“flip-flop”), and subsequent desorption of the FA into the cytosol. There still exists some dispute as to the identity of the rate-limiting step of FA transport. In the present study, sum-frequency vibrational spectroscopy (SFVS) was used to directly measure the rate of stearic acid (SA) flip-flop, in planar supported lipid bilayers (PSLBs) comprised of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The impact of SA on the physical properties of binary mixtures of SA and DSPC were investigated via -A isotherms from which the excess free energies of mixing and compression moduli were calculated. The manner in which these physical changes influenced the rates of SA and DSPC flip-flop were subsequently examined using SFVS. The rates of SA and DSPC flip-flop revealed that SA flip-flops independently of DSPC and on much faster timescales than its phospholipid counterpart. SFVS was also used to probe the rate of proteinunassisted SA desorption from hybrid supported lipid bilayers (HSLBs), allowing for the first decoupled measurement of the rates of desorption and flip-flop. These results provide strong evidence for desorption being the rate-limiting in FA transport through the membrane in the absence of proteins.

Introduction Fatty acids are the building blocks of phospholipids and an important energy source for cells. Fatty acids are composed of long hydrocarbon chains terminated in a carboxylic acid. In biological systems, these chains are unbranched, contain an even 2 ACS Paragon Plus Environment

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3 number of carbons and are either saturated or unsaturated.1 For unsaturated fatty acids, the cis-configuration is the most prevalent in nature. Some fatty acids cannot be synthesized by the human body, and thus must be obtained from food sources. These are referred to as “essential” fatty acids. The most common essential fatty acids are omega-3 and omega-6 fatty acids. In vivo, fatty acids are most commonly combined with glycerol to form triglycerides for storage and later use within cells. The lipolysis of these triglycerides is required to create free fatty acids, which can then be used in lipid synthesis. The endoplasmic reticulum and the Golgi apparatus are the sites were lipid biosynthesis occurs in cells.6 As a building block for phospholipids, free fatty acids are combined with either a sphingosine or glycerol backbone to produce phosphatidic acid (PA). PA is then transformed into the various lipid types by the addition of choline, ethanolamine, or serine to produce the lipids found in cellular membranes.2-5 Fatty acids are also important as a fuel source for adenosine triphosphate (ATP) generation.6 In order for fatty acids to be used in this capacity they must travel from the extracellular fluid, cross the plasma membrane, and pass into the cytoplasm and reach the mitochondria as free fatty acids. Since fatty acids are insoluble in water, they must bind to albumin in order to be transported extracellularly in plasma. The free fatty acid concentration in blood is therefore limited by the number of available albumin binding sites. Free fatty acid transport through the cellular membrane is thought to occur in three main steps: (1) adsorption of the fatty acid (FA) to the plasma membrane, (2) transmembrane movement (i.e. “flip-flop”), followed by (3) dissociation from the membrane into the cytoplasm; these steps are depicted in Figure 1.1, 7-8 One aspect of FA transport, which has been deliberated in the literature, is the

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4 identity of the rate-limiting step, which has come under such intense debate that some have termed the dispute the “fatty acid wars.”9 Several researchers have suggested that FA flip-flop is prohibitively slow such that proteins, namely fatty acid transport proteins (FATPs), are required to translocate FA across the membrane in order to meet the metabolic demands of the cell.8-12 The competing argument asserts that desorption of FAs from the plasma membrane into the cytosol is the slow step, implying that passive transmembrane movement of FAs is sufficient to sustain cellular metabolic requirements.10 There have been a number of techniques used to study FA transport, but because FAs have limited solubility in aqueous media, most studies to date have required the use of proteins such as bovine serum albumin (BSA), or vesicles loaded with FAs to deliver or extract FAs from the membrane in order to measure transmembrane movement. This presents an additional challenge in studying FA flip-flop, as factors related to FA-protein and FA-vesicle equilibria must also be considered. Careful studies on FA uptake and release via BSA have revealed affinity constants (Ka) of BSA for FAs and the rates of FA adsorption onto vesicle membranes.11 The affinity constants of BSA for fatty acid substrates has been determined to range from nM to M,11, 13 depending on fatty acid chain length and degree of unsaturation, while the measured rates of adsorption vary from 6 s-1 to. 0.04 s-1.1, 12, 14-16 Stopped-flow fluorescence spectroscopy has been one of the main techniques used to examine FA transport in phospholipid vesicles,1, 11-12, 15, 17-19 and the results from these studies have reported conflicting results regarding the rate-limiting step in FA transport. Cupp et al studied the kinetics of FA transfer between FA-loaded small


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5 unilamellar vesicles (SUVs) with larger unilamellar vesicles (LUVs) containing pyranine.11 Pyranine was used to detect the release of a proton from the FA upon reaching the inner leaflet of the vesicle by monitoring the reduction in fluorescence upon protonation of pyranine. The proton exchange between the FA and pyranine was assumed to occur quickly, therefore not limiting the measured dynamics. To measure FA desorption, SUVs were mixed with LUVs containing ADIFAB (acrylodan-labeled intestinal fatty acid binding protein) to detect the presence of free FAs that had desorbed from the inner leaflet. The authors of this study concluded that FA flip-flop was prohibitively slow based on the observed fast desorption of FAs from SUVs in the presence of ADIFAB.11 However, the ability of ADIFAB to extract FAs from the membrane and the rate of extraction were not measured. Competing evidence suggested that desorption is the rate-limiting step in FA transport. SUVs containing pyranine and fluorescein-phosphatidylethanolamine (FPE) were used to infer the rate of oleic acid (OA) flip-flop.15 FPE was used to detect the transfer of OA and also estimate the rate of OA flip-flop based on the measured decrease in FPE fluorescence.21 Because proton exchange between OA- and OA occurs quickly, it was assumed that OA- adsorbed onto the membrane, becomes protonated, and subsequently flips to the vesicle interior. From the fluorescence data and assuming rapid proton exchange, the authors determined an OA flip-flop rate of  69 s-1 at 22 ˚C. The measured OA flip-flop rate was determined to be 5-fold faster than the measured desorption rate of 3.4×10-3 s-1. Based on the measured rates of flip-flop and desorption, it was concluded that desorption was the rate-limiting step. These studies were complicated by the possibility of vesicle fusion, which could also be used to explain the observed



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6 results. The size of the vesicles in the mixed solution was not measured to test this possibility. As the examples above illustrate, the controversy regarding the rate limiting step in FA transport stems mainly from the inability to measure FA flip-flop and desorption rates directly without the use of an exogenous reporter, such as pyranine, FPE or ADIFAB or the use of a FA delivery agent such as BSA or lipid vesicles. Nonlinear optical methods are an alternative to linear spectroscopic techniques for measuring molecular transport through membranes. For example, the nonlinear optical technique of second harmonic scattering (SHS) has been used to measure the transport of the dye malachite green (MG) through lipid vesicle and bacterial membranes, providing unprecedented insight into the molecular transport mechanism (adsorption, translocation, and desorption) in biological membranes.20-25 These studies are remarkable in that they directly measured the kinetics of molecular transport through a lipid membrane without the need for an exogenous reporter by taking advantage of the intrinsically second harmonic (SH) active dye MG.24-25 These studies were possible due to the unique symmetry aspects of a second-order nonlinear optical process, which negates the signal from a symmetric system, such as when the same number of molecules reside on both sides of a membrane. Measuring FA transport using SHS is problematic due to the lack of an accessible electronic transition, though attempts have been made to infer the transport of ionic species, across lipid membranes in vesicles and the outer-membrane protein channels in living bacteria using MG as the reporter.22, 26 In this paper, we present a new method for directly and separately measuring FA transport (flip-flop and desorption) kinetics in a phospholipid bilayer using the companion nonlinear method of sumfrequency vibrational spectroscopy (SFVS).


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7 SFVS is a coherent, nonlinear, second-order optical technique that involves the spatial and temporal overlap of a coherent tunable infrared (IR) and fixed visible (VIS) light sources at an interface between two isotropic media, whereby a photon is generated at the sum of the two incident frequencies (SUM): ω𝑆𝐹 = ω𝑉𝑖𝑠 + ω𝐼𝑅,

(1)

where ω denotes frequency, and subscripts SF, Vis, and IR describe the resultant sumfrequency, visible and infrared sources, respectively. The intensity of the sum-frequency response is described by:

|

𝑁⟨𝑀𝑖𝑗𝐴𝑘⟩

|

2 𝐼𝑆𝐹 ∝ |𝜒(2)| ∝ ∑𝜈𝜔𝜈 ― 𝜔𝐼𝑅 ― 𝑖𝛤𝜈

2

(2)

where χ(2) is the first-order nonlinear susceptibility tensor, N is the number of molecules interacting with the incident electric fields, Mij is the Raman transition probability, Ak is the IR transition probability, ων is the frequency of the ν vibrational mode, ωIR is the frequency of the tunable IR, and Γ is the linewidth of the resonant vibrational state. The angular brackets denote an average over all orientations.27 Our group has previously shown that SFVS can be used to directly measure native lipid translocation in planar supported lipid bilayers (PSLBs).28-34 As with SHS, the coherent nature of SFVS results in destructive interference of the normal components of the emitted sum-frequency signals from the two leaflets of a symmetric bilayer, due to their antiparallel orientation. However, by selectively deuterating one of the leaflets, the frequency shift in the vibrational modes allows one to eliminate the destructive interference between the two leaflets. Using the CH3 symmetric stretch (vs) intensity from the termini of the lipid acyl chains as an indicator of membrane asymmetry, the number 7 ACS Paragon Plus Environment


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8 difference of proteated lipids between the proximal (NPROX) and distal (NDIST) leaflets in a PSLB, is given by the expression: 𝐼𝐶𝐻3𝑣𝑠 ∝ (𝑁𝑃𝑅𝑂𝑋 ― 𝑁𝐷𝐼𝑆𝑇)2.

(3)

As lipids flip-flop between leaflets, the intensity of the CH3 S decreases and eventually reaches a minimum, indicating that the number of proteated lipids in the proximal and distal leaflets is equal (i.e. NPROX=NDIST). The rate of lipid flip-flop is measured by monitoring the symmetric CH3 S over time and fitting the resulting decay to the expression: 𝐼𝐶𝐻3(𝑡) = 𝐼𝑚𝑖𝑛 + 𝐼𝑚𝑎𝑥𝑒 ―4𝑘𝑡

(4)

where Imax is the initial CH3 S intensity of the as prepared asymmetric bilayer, k is the flip-flop rate constant, and Imin is the signal offset of the detection system.57 The CH3 S stretch is measured using the ssum-frequecy, svis, pIR polarization combination to probe the transition dipole moments along the surface normal of the membrane.57 The studies presented herein were motivated by the need to directly measure the rates of native FA flip-flop and desorption in order to determine the rate limiting step to FA transport. Stearic acid (SA) in a PSLB of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) prepared by the Langmuir-Blodget/Langmuir-Schaefer (LB/LS) method was used as the model system. We have previously shown that membrane properties such as lipid packing and the molar compression modulus are strongly correlated to the rate of lipid flip-flip.33,

35-36

Membrane packing of the binary SA and DSPC membranes was

characterized by measuring pressure ()-area (A) isotherms, which were then used to quantify the energetics of mixing. SA:DSPC membranes with selectively proteated SA or DSPC were prepared in order to directly measure the transmembrane movement of the 8 ACS Paragon Plus Environment

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9 proteated species. Hybrid supported lipid bilayers (HSLBs) were also prepared in order to independently measure SA desorption. Combined, these studies have provided the first ever decoupled measurements of the rates of FA translocation and protein-free desorption. The results from these studies suggest that, in the absence of any proteins, desorption is the rate-limiting step to fatty acid transport through a lipid membrane.

Experimental Methods Materials. All materials were used as received unless otherwise noted. 1,2-distearoyl-sn3-glycero-phosphocholine (DSPC) and 1,2-distearoyl-d70-sn-3-glycero-phosphocholine (DSPCd70) were purchased from Avanti Polar Lipids (Alabaster, AL). Stearic acid (SA), stearic acid-18,18,18-d3 (SAd3), methyltriethoxysilane (MTES), chloroform, and deuterium oxide were purchased from Sigma Aldrich Millipore (St. Louis, MO). Potassium

phosphate

anhydrous

(dibasic),

potassium

phosphate

monohydrate

(monobasic), toluene, molecular sieves, and hydrogen peroxide were purchased from Fisher Scientific (Pittsburgh, PA). Toluene was dried with molecular sieves for at least 24 hours prior to use. Sodium chloride, NaOH pellets, H2SO4, and HCl was purchased from Macron Chemicals (Center Valley, PA). The water used in the studies had a minimum resistivity of 18.2 MΩ•cm and was obtained from a Barnstead Thermolyne, (Dubuque, IA) Nanopure™ water system. 150 mM phosphate buffered saline (PBS) was prepared from 100 mM NaCl, 10 mM NaH2PO4•H2O, and 40 mM Na2HPO4 in Nanopure water and adjusted to a final pH of 7.4 ± 0.1 using 4 M NaOH and 1 M HCl. UV-IR grade fused silica prisms purchased from Almaz Optics (Marlton, NJ) were used as bilayer supports.



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10 Solutions of SA, SAd3, DSPC, and DSPCd70 were prepared at a concentration of 1 mg/mL in chloroform. These stock solutions were used to prepare the 1:7, 1:3, and 1:1 SA:DSPC membranes. The selection of the proteated component depended on the species of interest for a given experiment, either SA or DSPC. For example, for a measurement of the SA component in a 1:1 SA:DSPC membrane, a bilayer of SAd3:DSPCd70 (proximal) and SA:DSPCd70 (distal) was prepared. To study the DSPC component in a 1:1 SA:DSPC membrane, a bilayer of 1:1 SAd3:DSPCd70 (proximal) and 1:1 SAd3:DSPC (distal) was prepared. The proteated component was incorporated in the distal leaflet for consistency. PSLBs with the reverse deposition order were prepared in order to verify that the order of preparation did not influence the measured rates of flip-flop. Pressure()-Area(A) Isotherms. Pressure-area isotherms were collected on a Langmuir Trough (KSV, Helsinki, Finland). Stock solutions of the SA:DSPC mixtures in chloroform were spread at the air-water interface with PBS as the subphase. The lipid monolayer was allowed to equilibrate for 15 minutes prior to compression to ensure evaporation of the solvent. The SA:DSPC monolayers were compressed at a rate of 4 mm/min at a temperature of 22 ± 0.1 ºC. The average of 3 isotherms were taken. Bilayer Preparation by the LB/LS Method. Model membranes were prepared on clean SiO2 prisms by the Langmuir-Blodget/Langmuir-Schaeffer (LB/LS) method. 34, 37 Prior to use, prisms were placed in an UV-ozone cleaner (Jetlight Co., Irvine, CA) for 30 minutes. Afterwards, the prisms were immersed in piranha solution prepared with 70% 18 M sulfuric acid and 30% H2O2 for a minimum of 30 minutes. (Caution: This is a highly corrosive solution and a strong oxidant that reacts violently with organic solvents. Extreme caution must be taken when handling piranha solution.) The prisms were then


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11 rinsed with Nanopure water, dried under nitrogen, and treated with an argon plasma (Harrick Scientific, Ithaca, NY) for two minutes. A clean fused SiO2 support was submerged in the PBS subphase of the LB trough. The SA:DSPC mixtures in chloroform were then deposited at the air-water interface and allowed to equilibrate for 15 minutes to ensure complete solvent evaporation. The monolayers were compressed to a surface pressure of 30 mN/m at a rate of 4 mm/min. A surface pressure of 30 mN/m was chosen as this reflects the pressure of the plasma membrane in cells and solution phase vesicles (30-35 mN/m).

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The prism was then withdrawn vertically through the compressed

monolayer, depositing the proximal leaflet onto the prism surface via a LB transfer. Afterwards, the subphase was removed, and the trough was washed thoroughly with water, isopropanol, and methanol. The PBS subphase was replaced in the trough, and the desired SA:DSPC mixture for the distal leaflet was deposited on the subphase. After compressing to a final surface pressure of 30 mN/m, the prism with the LB layer was rotated horizontally and submerged through the compressed monolayer to form the distal leaflet by a LS transfer. The prism was then transferred to a custom-built Teflon flow cell while constantly under an aqueous environment. Afterwards, the solution in the flow cell was replaced with PBS in D2O to avoid spectral interference from H2O in the C-H region. Hybrid Supported Lipid Bilayers (HSLBs). The procedure for preparing HSLBs is described in detail elsewhere, but the salient steps are discussed here.39-40 Cleaned fused SiO2 prisms were submerged in a solution of 2% (v/v) MTES in dry toluene for approximately 16 hours. The prisms were then thoroughly rinsed with chloroform and dry toluene. Afterwards, LS deposition of the 1:7, 1:3, or 1:1 SA:DSPCd70 layer was deposited using the LS transfer method as described above.



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12 SA and DSPC Flip-Flop Kinetics. The spectrometer setup used for these studies has been detailed in elsewhere. Prior to kinetics measurements, vibrational spectra were collected from 2750 to 3050 cm-1 in 2 cm-1 steps and integrating for 4 s at each step. To obtain the rates of SA or DSPC flip-flop, the CH3 νs was monitored continuously as a function of time and was averaged in 4 s intervals. All spectra and kinetic data were recorded using the ssp polarization combination.30 To regulate the temperature, a circulating water bath (HAAKE, Phoenix II P1 Circulator, Thermo Fischer Scientific) was connected to the flow cell which was also equipped with a type K thermocouple to record the temperature. Results & Discussion -A Isotherms. The effect of SA on DSPC packing was evaluated from the -A isotherms of mixed monolayers of SA and DSPC (Figure 2). The measured mean molecular areas (MMAs) at 30 mN/m for monolayers of pure SA, 1:7 SA:DSPC, 1:3 SA: DSPC, 1:1 SA:DSPC, and pure DSPC were 19 ± 2 Å2/molecule, 28.0 ± 0.3 Å2/molecule, 32.3 ± 0.5 Å2/molecule, 40.9 ± 0.9 Å2/molecule, and 46 ± 0.1 Å2/molecule, respectively. The experimentally measured MMA of DSPC at 30 mN/m agrees well with previously reported values.33-34,

41

From the isotherms, the MMA of the SA:DSPC monolayer

decreased as the relative amount of SA increased in the monolayer. To determine if attractive or repulsive forces were present between SA and DSPC, the experimentally determined MMAs were compared with the theoretical MMAs for an ideal mixture, which were calculated using the weighted average of the MMAs for the two components:42 A12 = A1X1 + A2X2,

(5)


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13 where A12 is the theoretical MMA, A1 and A2 are the mean molecular areas of components 1 and 2, respectively, and X1 and X2 are the mol fractions of each component (Figure 4). The SA:DSPC mixtures exhibit MMAs smaller than what is predicted by eq. 5, which indicates an attractive interaction between SA and DSPC. This condensation of the membrane has been previously observed in mixtures of palmitic acid (PA) and 1,2dipalmitoyl-sn-glycero-3-phoshocholine (DPPC) as well as SA:DPPC mixtures.43-44 Excess Free Energy of Mixing. To quantify the attractive forces between SA and DSPC and their influence on SA transport, the excess free energy of mixing (Gexc) was calculated for the various binary mixtures using the following expression:42 Π

𝛥𝐺𝑒𝑥𝑐 = 𝑁∫0 𝐶(𝐴12 ― 𝐴1𝑋1 ― 𝐴2𝑋2)𝑑𝛱,

(6)

where N is Avogadro’s number, and C is the monolayer collapse pressure. The calculated Gexcs are plotted as a function of mol% SA in Figure 5. For the SA:DSPC mixtures, the negative Gexc indicates favorable mixing of the components due to attractive interactions between SA and DSPC. Negative Gexc values of SA:DPPC mixtures were also reported by Hac-Wydro et al.44 Interestingly, our data suggest that the most energetically favorable mixing occurs in the 1:3 SA:DSPC mixture, while HacWydro et al observe this for the 1:1 SA:DPPC mixture for surface pressures up to 25 mN/m. Ma et al have also observed the most favorable mixing in a 1:1 palmitic acid (PA):DPPC mixture, though this was reported for DPPC in the LE/LC phase (=12 mN/m).43 It is likely that the discrepancy between the values reported here and previous studies is due to the ionic strength of the subphase. Ma37 and Hac-Wydro38,39 et al prepared mixed monolayers of fatty acids and phospholipids on a neat water subphase, while the studies herein used a 150 mM PBS subphase at pH 7.4 to reflect physiological 13 ACS Paragon Plus Environment


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14 conditions. Previous calculations of the ΔGexc of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and oleic acid triglyceride mixtures on both neat water and 0.55 M NaCl showed that the ionic strength of the subphase played a significant role in shaping the magnitude of ΔGexc,45 with the magnitude of ΔGexc

increasing with

increasing ionic strength. Compression Modulus. The compression modulus (K) was also calculated from the A isotherms to examine the impact of SA on the monolayer compressibility. The compression modulus is defined as:41 K = –A(dΠ/dA).

(7)

A numerical derivative of the -A isotherms was performed (Figure 2) using the following expression:41 K(Πi) = –Ai(Πi+1 – Πi-1)/(Ai+1 – Ai-1),

(8)

where i+1 and i-1 indicate the interval of the running numerical derivative, with the calculated compression moduli as a function of surface pressure presented in Figure 5. Abrupt changes in the compression modulus are indicative of a phase transition, as seen for a pure SA monolayer at ~23 mN/m.46 For the 1:7 SA:DSPC, 1:3 SA:DSPC, 1:1 SA:DSPC, and pure DSPC monolayers, the shape of the compression modulus curves indicates that the monolayers are within a single phase between 10 to 45 mN/m.47-48 The calculated compression moduli at 30 mN/m for pure DSPC, 1:7 SA:DSPC, 1:3 SA: DSPC, 1:1 SA:DSPC, and pure SA are 270 ± 20, 280 ± 3 mN/m, 260 ± 3 mN/m, 205 ± 3 mN/m, and 860 ± 50 mN/m, respectively. The experimentally determined modulus of 270 ± 20 for a pure DSPC monolayer at 30 mN/m is consistent with the previously determined modulus of 270-300 mN/m at 30 mN/m.33, 41, 49 As the mol% of SA increased 14 ACS Paragon Plus Environment

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15 in the monolayer, the compression moduli decreased, indicating that the membrane became more compressible. Previously determined compression moduli of SA:DSPC monolayers over a neat water subphase showed that the compression modulus did not change appreciably between 10-30 mol% SA.47

However, at 50 mol% SA the

compression modulus increased to roughly 400 mN/m, which is in contrast to the observed decrease to 205 ± 3 mN/m. Similar to the discrepancies in MMA discussed above, the conflicting compression modulus of the 1:1 SA:DSPC mixture is most likely due to the differences in ionic strength of the subphase used in the two experiments. It has been previously shown that the compression modulus of DPPC decreases when the subphase is changed from neat water to 0.15 M buffer (HEPES, NaCl), as observed here.50 SA and DSPC Flip-Flop Kinetics. Bilayers with selectively deuterated and proteated components were prepared such that a sum-frequency response would arise from only proteated SA or DSPC (Figure 6). For consistency, the proximal leaflet was always composed of SAd3:DSPCd70, while the distal leaflet was composed of SAd3:DSPC or SA:DSPCd70 to monitor the DSPC (Figure 6A) or SA (Figure 6B) components, respectively. The SFVS spectra of DSPC show the characteristic acyl chain vibrational modes at 2850 cm-1 (CH2 S), 2875 cm-1 (CH3 S), 2898 cm-1 (CH2 AS), 2936 cm-1 (CH3 FR), and 2960 cm-1 (CH3 AS). For the SA spectra, Figure 6B, the spectra only display the characteristic CH3 resonances as only the terminal CH3 is deuterated (CD3) and not the entire acyl chain. Due to the coherent nature of SFVS, the CH2 resonances from SA and SAd3 in the opposing leaflet destructively interfere.

The amplitude of the

characteristic resonances of SA and DSPC did not change as a function of SA content in



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16 the membrane, determined by fitting the spectra to eq. 2. The SFVS spectra were used to confirm the presence and quality of the asymmetric PSLBs prior to kinetics measurements. The rates of SA flip-flop in the 1:7, 1:3, and 1:1 SA:DSPCd70 PSLBs were measured over a range of temperatures. For comparison, the rates of flip-flop of a pure SA:SAd3 membrane was measured as a baseline. Representative decay curves for SA flip-flop at 20˚C are presented in Figure 7 and the measured rates and calculated halflives are listed in Table 1. The half-life (t1/2) of flip-flop was determined using eq. 9: 𝑡1/2 =

ln (2) 2𝑘

(9)

In a SA:SAd3 PSLB, the half-life of flip-flop at 20 ˚C was 28.6 ± 0.5 minutes. At the same temperature, the half-lives of flip-flop in the 1:1 and 1:7 SA:DSPCd70 mixtures were 53 ± 2 and 83.4 ± 0.9 minutes, respectively, while the half-life of flip-flop of the 1:3 SA:DSPCd70 mixture was slightly faster at 21.2 ± 0.2 minutes. In order to rule out potential interactions between the bilayer and solid support, an additional bilayer was prepared with SA in the proximal leaflet as a control, and the rate of SA flip-flop was measured near 20 ˚C (Figure 8). The measured SA flip-flop rates in the proximal and distal leaflets were (8.37 ± 0.08)×10-5 s-1 and (8.19 ± 0.07)×10-5 s-1, respectively, at 20 ˚C, where the rate of SA flip-flop in the distal leaflet was extrapolated from the data in Table 1. The rates correspond to half-lives of 69.0 ± 0.7 minutes and 70.1 ± 0.7 minutes, respectively. The agreement between the flip-flop rates demonstrates that the order of deposition does not significantly impact the measured rates, suggesting that SA does not have any significant interactions with the solid support. We have previously shown that the order of deposition had no effect on the measured rates of DSPC flip-flop.46


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17 SA flip-flop in egg PC large unilamellar vesicles (LUVs) was previously measured by stopped-flow fluorescence spectroscopy and determined to be ~23 ms-1 at 25 ˚C.19 Using the same technique, the flip-flop rate of OA was determined to be ~5 s-1 in reconstituted plasma membranes (PMs) at 37 ˚C.51 Based on the data from Table 1, the extrapolated rates of SA flip-flop at 25 ˚C of the 1:0, 1:7, 1:3, and 1:1 SA:DSPCd70 mixtures would be 0.4 ± 0.6 ms-1, 0.19 ± 0.06 ms-1, 1.0 ± 0.8 ms-1, and 0.2 ± 0.2 ms-1, respectively. The differences in the extrapolated rates and reported rate of SA flip-flop could be due to the phospholipid matrix: DSPC is in the gel phase, while egg PC is comprised of mostly POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and is in the fluid phase at 25 ˚C.52-53

Nevertheless, the measured rates of SA flip-flop are

consistent within an order of magnitude, which is remarkable given the differences in the composition of the membranes. The rate of SA flip-flop in the SA:DSPC mixtures is fastest for the 1:3 SA:DSPC membrane, which is also faster than in a neat SA bilayer, while the rates of flip-flop decrease slightly for the 1:1 and 1:7 mixtures. The rates do not appear to be correlated with changes in the average MMA of the monolayer (Figure 3). However, the rate of SA flip-flop does seem to correlate with the free energy of mixing between SA and DSPC, Figure 4. A comparison of the SA flip-flop rate data as a function of SA content and Gexc suggests that the more favorable the interactions between SA and DSPC, the faster the rate of SA translocation. For example, the 1:3 SA:DSPC membrane has the fasted rate of SA flip-flop and the largest Gexc at -1.27 ± 0.08 kJ/mol. The 1:7 and 1:1 SA:DSPC membranes have Gexc of -0.35 ± 0.6 kJ/mol and -0.78 ± 0.03 kJ/mol and correspondingly slower rates of flip-flop.



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18 It is generally accepted that FAs flip-flops in the unionized form (FA) instead of the ionized state (FA-).1, 12 However, SFVS is not capable of distinguish between the two forms. It has been previously shown that the addition of OA micelles to pyranine-loaded vesicles resulted in a fast decrease in pH in the interior of the vesicle.54 The fast decrease could only be attributed to fast flip-flop of OA, which shuttled protons to the vesicle interior. Additionally, MD simulations on OA flip-flop showed that the energetic barrier to OA- flip-flop through the center of the membrane was much higher than OA. The discrepancy in the energy barrier between OA and OA- flip-flop was large enough that the authors ignored the contribution of OA- to flip-flop.55 In light of the reported studies on the ionization state of OA, it is likely that the measured SA flip-flop rates correspond to the unionized SA instead of SA-. In addition to measuring SA flip-flop kinetics, the effect of SA on DSPC flip-flop was also measured and presented in Figure 9. The effect of introducing FAs on phospholipid flip-flop has remained relatively unexplored.56 A prior study monitored the influence of docosahexanoic acid (DHA) on flip-flop of NBD-PE (1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,2-benzoxadazol-4-yl)) vesicles. These results suggested that DHA, a polyunsaturated fatty acid, increased phospholipid flipflop. Representative decays obtained by measuring the CH3 S intensity from DSPC over time for PSLBs of 0:1, 1:7, 1:3, and 1:1 SAd3:DSPC at 46 ˚C are presented in Figure 9. The measured half-life (eq. 2.16) of DSPC flip-flop in a pure DSPC membrane at 46 ˚C was 86.0 ± 0.1 minutes, which is in good agreement with previously measured values of DSPC flip-flop.57-58 This rate is much faster in comparison to the half-lives of DSPC flipflop at 46oC for the 1:7, 1:3, and 1:1 SAd3:DSPC mixtures, which were determined to be


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19 791 ± 3 minutes, 223 ± 2 minutes, and 465 ± 2 minutes, respectively. The presence of SA appears to slow down flip-flop of the DSPC component in the membrane (Figure 9). It has been previously shown in PSLBs of DSPC that tighter membrane packing (i.e. smaller MMAs) results in a decrease in the flip-flop rate.28 To account for the differences in the MMAs of DSPC in the mixed bilayers, the theoretical rates of DSPC flip-flop were calculated using the measured MMAs from Figure 3, scaled by the mol% of DSPC in the membrane using eq. 5. Based on the high compression modulus of SA at 30 mN/m, it was assumed that the MMA of SA does not change significantly in the SA:DSPC mixtures. Using a fixed MMA of 19 Å2/molecule for SA, the calculated MMAs for DSPC in the 1:7, 1:3, and 1:1 SA:DSPC mixtures were 43.3 Å2/molecule, 36.9 Å2/molecule, and 37.0 Å2/molecule, respectively. Using the previously determined relationship between DSPC flip-flop and membrane packing,28 the theoretical rates of DSPC flip-flop at 46 °C were computed to be (3.2 ± 0.3)x10-5 s-1, (2.1 ± 0.2)x10-5 s-1, and (2.1 ± 0.2)x10-5 s-1 in the 1:7, 1:3, and 1:1 SAd3:DSPC PSLBs, respectively. Using the measured rates from Table 1, the extrapolated rates of DSPC flip-flop in the 1:7, 1:3, and 1:1 SAd3:DSPC were (5.7 ± 0.2)x10-6 s-1, (2.1 ± 0.1)x10-5 s1, and (1.1 ± 0.2)x10-5 s-1 respectively, at 46 ˚C. A comparison of the measured and calculated rates shows that the theoretical rates of DSPC flip-flop were 5.6 times, 1.0 times, and 2.0 times faster than the measured rates in the 1:7, 1:3, and 1:1 SAd3:DSPC PSLBs, respectively. Because the theoretical and measured rates are different within only an order of magnitude, this comparison suggests that the measured decrease in the rates of DSPC flip-flop can be largely explained by the changes in membrane packing. A possible explanation for the slight increase in the rate of DSPC flip-flop



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20 compared to the calculated rate based on MMA is an interaction between SA and DSPC which facilitates translocation. Previous SFVS spectra of monolayers of PA (palmitic acid) and DPPCd62 (1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine) showed that the PO2- S stretch was red shifted in all PA:DPPCd62 mixtures.43 The red-shift was attributed to a hydrogen bond between the phosphate backbone of the PC headgroup and the neutral carboxylic acid group of PA. The observed discrepancy in the DSPC flip-flop kinetics could be due to the presence of a hydrogen bond interaction between SA and DSPC. If a strong hydrogen bonding interaction is present, the rapid SA flip-flop could assist DSPC flip-flop in the membrane. A comparison of the measured rates of SA and DSPC flip-flop reveal that SA and DSPC flip-flop occur on vastly different time scales. For instance, the extrapolated halflife of DSPC flip-flop at 23 ˚C for the 1:0, 1:7, 1:3, and 1:1 SAd3:DSPC mixtures is 252 ± 6 hours, 1310 ± 80 hours, 68 ± 20 hours, and 60 ± 10 hours, respectively. These halflives are 940, 190, and 68 times slower than SA flip-flop in the 1:7, 1:3, and 1:1 SA:DSPC PSLBs at 23 ˚C, respectively. The faster rates of SA flip-flop could be attributed to its smaller size. Previous kinetics measurements of phospholipid flip-flop have shown that larger headgroups, such as TEMPO (TEMPO=2′,2′,6′,6′-tetramethyl-4′piperidyl)-DPPC, slows flip-flop.57 Thus, a smaller headgroup, such as the neutral carboxylic acid group in SA, should result in faster flip-flop. In addition, to the headgroup, the fact that SA is composed of only a single acyl chain compared to DSPC, an increase in the rate of translocation is also expected. We have previously shown that cholesterol (CHO), a small amphiphilic molecule, had a rate of translocation too fast to measure in a DSPC membrane.57 The decoupled nature of SA and DSPC flip-flop might


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21 suggest phase segregation. If this were true, DSPC and SA would form distinct domains that could flip-flop independently of each other, leading to two different flip-flop rates. However, the compression moduli (Figure 5) show that the SA:DSPC mixtures are within a single phase and do not exhibit phase segregation. Furthermore, Brewster Angle Microscope (BAM) images of SA:DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) membranes show no phase segregation at 35 mN/m at 20 ˚C.59 Taken together, the compression moduli and BAM images suggest that phase segregation was not occurring in the SA:DSPC model membranes. In addition, if there was phase segregation, the DSPC component would have a larger MMA and rate comparable to that for a pure DSPC membrane, as was observed in binary mixtures of CHO+DSPC.58 Transition State Thermodynamics. Using the measured flip-flop rates of SA and DSPC, the free energies of activation (G‡) were calculated using the Eyring equation: ∆𝐺 ‡ = ―𝑅𝑇𝑙𝑛

( ) 𝑘ℎ 𝑘𝐵𝑇

(10)

where k is the measured rate of SA or DSCP flip-flop, kB is Boltzmann’s constant, h is Planck’s constant, R is the ideal gas constant, and T is the temperature in Kelvin. The calculated free energies of SA and DSPC flip-flop as a function of temperature are presented in Figure 11, and the activation free energies at 22˚C are listed in Table 2. The activation free energy barrier represents the energy difference between the ground and transition state geometries of the lipid undergoing flip-flop. Factors that govern the magnitude of the activation free energy barrier include lipid-lipid interactions (e.g. hydrogen bonds, van der Waals forces) and also lipid-solvent interactions.33 A comparison of the activation free energies of SA and DSPC flip-flop (Table 2) reveals that the G‡ of SA flip-flop is much lower than DSPC at 22 ˚C (295 K) for all the 21 ACS Paragon Plus Environment


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22 SA:DSPC mixtures. The decrease in G‡ could be due to the difference in size between SA and DSPC, where the neutral carboxylic acid headgroup of SA could introduce less of an energetic penalty to translocation across the hydrophobic membrane core than the larger zwitterionic headgroup of DSPC. We have shown previously that changes in headgroup size, such as in TEMPO-DPPC, increased the G‡ of flip-flop.57 In addition to the headgroup differences, the fact that SA only has one acyl chain, versus the two acyl chains in DSPC, could also lower the free energy barrier to SA flip-flop. A comparison of the free energy of mixing and the free energy of flip-flop reveals that the G‡ for SA flip-flop in the SA:DSPC mixtures decreases as the absolute magnitude of Gexc (Table 2), increases. Because Gexc depends on the favorable interactions between SA and DSPC, the data suggest that the favorable mixing within the monolayer results in favorable mixing between leaflets. In mixed monolayers of DSPC and cholesterol (CHO), 0-20 mol% CHO was found to condense these monolayers at 25°C in the solid-ordered (so) phase, and the Gexc of these CHO:DSPC mixtures were negative and favorable.60-61 Separately, in model membranes of DSPC and CHO in the same concentration range, it was shown by SFVS that the presence of cholesterol lowered the free energy barrier to DSPC flip-flop.58 Taken together, these studies suggest that favorable mixing between DSPC and CHO resulted in an increase in Gexc, which was coupled to a decrease in the G‡ of DSPC flip-flop. This behavior is similar to the observed relationship between G‡ and Gexc for SA, observed here. The transition state enthalpy (‡) and entropy (S‡) for SA and DSPC flip-flop were determined from the activation free energies as a function of temperature using the Gibbs equation (eq. 11): 22 ACS Paragon Plus Environment

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23 ∆𝐺 ‡ = ∆𝐻 ‡ ―𝑇∆𝑆 ‡

(11)

Examination of H‡ and S‡ reveals that they are linearly correlated (Figure 12), indicative of strong enthalpy-entropy compensation. The degree to which the entropy compensates for the enthalpy is described by: ∆𝐻 ‡ = ∆𝐻 ‡ , ∗ + 𝑇𝑐∆𝑆 ‡

(12)

where Tc is the compensation temperature, and H‡,* the compensation enthalpy.3 The compensation temperature for SA and DSPC is 285 K. This value is significantly different from the average experimental temperature over which the data were collected, 308 K, which suggests the observed correlation is not due solely to error propagation.62-63 The observation that the data for both SA and DSPC follow the same trend in enthalpyentropy compensation suggests that a common mechanism is controlling the flip-flop of the two species. This is remarkable considering the chemical difference between SA and DSPC and their varied concentrations in the membrane. It has previously been observed that values of Tc in the range of 270-300 K have been linked to water solvation playing a dominate role in the reaction thermodynamics of a variety of process, from protein folding to protein-ligand interactions and micelle formation.1,23 Based on the calculated Tc, one plausible explanation for the similarity in enthalpy-entropy compensation for both SA and DSPC is that solvation/de-solvation drives the observed entropy-enthalpy compensation.64 The dominate role of solvent re-organization is also apparent by the positive values of H‡ as a function of S‡, suggesting an entropy driven process in the temperature range examined.65 SA Desorption. The controversy over fatty acid transport centers on whether flip-flop or desorption is the rate-limiting step. The measured SA flip-flop rates determined by SFVS



Page 24 of 45

24 were roughly similar to reported rates of SA flip-flop determined by various stop-flow fluorescence measurements.14, 19, 66 Also, the rate of SA flip-flop was determined to be dramatically faster than DSPC flip-flop at room temperature. As with some previous studies, the SFVS results suggest that SA flip-flop is fast relative to lipid translocation, but in order to determine the rate limiting step in FA transport, independent desorption experiments were conducted. From SFVS, the rates of SA flip-flop were determined to be fast, but this alone does not identify the rate-limiting step in FA transport. There is also the possibility that the measured CH3 s decays for SA reported above were due to both flip-flop and desorption, as SFVS of the SA:DSPC PSLBs would not be able to distinguish between these two processes. To address both of these points, a separate measurement was performed in order to decouple SA flip-flop and desorption. HSLBs comprised of only a single SA:DSPCd70 leaflet in contact with a hydrophobic surface were prepared (Figure 14). HSLBs eliminated the possibility of SA flip-flop, providing a means to assess the desorption of SA independently. The peaks at 2910 cm-1 and 2970 cm-1 correspond to the the CH3 S and CH3 FR, respectively, of the surface Si-CH3 groups (Figure 14B).40 The measured CH3 S intensity from SA as a function time is plotted in Figure 15 for a 1:1, 1:3, and 1:7 SA:DSPCd70 HSLB. Over the course of 10 hour, no desorption of SA was observed for any of the SA:DSPCd70 HSLBs examined. A comparison of the relative time-scale of the of desorption experiment and SA flip-flop rates at 20 ˚C demonstrate that desorption does not contribute to the measured SA flip-flop rates. More importantly, these results also provide strong evidence for protein-unassited desorption being the ratelimiting step in FA transport. Our results are consistent with previous investigations that


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25 conclude that FA flip-flop is faster than desorption.1, 14, 17, 19 However, previous studies utalizing BSA have determined dissociation rates on the order of seconds.11, 14 Yet, the contribution due to BSA on the desoprtion rate was not examined. As described previously, a protein-free experiment using doubly-labeled pyranine and FPE SUVs was used to probe the desorption of OA.14 The fluorescence profile over time was used to determine a t1/2 of desorption of 100 ms. However, this rate could also be due to intervesicle transfer of OA and not represent the true rate of desorption of OA into solution. Our measurements remove the added complexity of using albumin and intervesicle transfer and provides the first direct measurement of SA protein-free “desorption” from a lipid membrane. Based on the relative rates of SA flip-flop and desorption, our results present strong evidence for desorption as the rate-limiting step in FA transport through a lipid membrane in the absence of a carrier protein such as albumin. Conclusions Model SA:DSPC mixed bilayers were studied by SFVS in order to characterize SA flip-flop in the context of identifying the rate limiting step in FA transport. We have shown from the -A isotherms that SA:DSPC mixtures exhibit tighter membrane packing than what would be predicted for an ideal mixture, indicative of attractive forces between SA and DSPC. The compression moduli of the SA:DSPC mixtures demonstrated that increasing the mol% of SA in the monolayer subsequently decreased the compressibility of the monolayer. The presence of SA had significant impacts on the physical properties of the membrane, which generally agreed with previous findings. As the dispute over FA transport has focused on whether flip-flop is relatively slower or



Page 26 of 45

26 faster than desorption, the flip-flop rates of SA were directly measured using SFVS. The rates demonstrated that SA flip-flop occurs quickly and agreed with previous measurements of FA flip-flop in vesicles. The effect of SA on DSPC flip-flop was also investigated by SFVS, which showed that DSPC flip-flop was apparently slower in the presence of SA in all SA:DSPC membranes studied. However, it was found that the changes in the rates of DSPC in the SA:DSPC PSLBs could be explained by changes in membrane packing. Finally, desorption rates of SA from HSLBs were directly obtained by SFVS, and provided a means to decouple SA flip-flop from desorption. A comparison of the relative rates of SA flip-flop and desorption provided strong evidence for proteinunassisted desorption as the rate limiting step in FA transport. Acknowledgments The authors would like to thank the National Science Foundation for their financial support (#1608550). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.


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27 References 1. Hamilton, J. A., Fatty Acid Transport: Difficult or Easy? J Lipid Res 1998, 39, 467-381. 2. Lehninger, A. L.; Nelson, D. L.; Cox, M. M., Principles of Biochemistry. 2008; p 372-372. 3. Athenstaedt, K.; Daum, G., Phosphatidic Acid, a Key Intermediate in Lipid Metabolism. Eur. J. Biochem 1999, 266 (1), 1-16. 4. Carman, G. M.; Han, G.-S., Phosphatidic Acid Phosphatase, A Key Enzyme in the Regulation of Lipid Synthesis. J Biol Chem 2009, 284 (5), 2593-2597. 5. Loewen, C. J. R.; Gazpar, M. L.; Jesch, S. A.; Delon, C.; Ktistakis, N. T.; Henry, S. A.; Levine, T. P., Phospholipid Metabolism Regulated by a Transcription Factor Sensing Phosphatidic Acid. Science 2004, 304 (5677), 1644-1647. 6. Lehninger, A. L.; Nelson, D. L.; Cox, M. M., Principles of Biochemistry. 2008. 7. Abumrad, N.; Harmon, C.; Ibrahimi, A., Membrane transport of long-chain fatty acids: evidence for a facilitated process. J Lipid Res 1998, 39, 2309-2318. 8. Schaffer, J. E., Fatty acid transport: the roads taken. American Journal of Physiology-Endocrinology and Metabolism 2015, 282 (2), E239-E246. 9. Pownall, H.; Moore, K., Commentary on fatty acid wars: The diffusionists versus the translocatists. Arteriosclerosis, Thrombosis, and Vascular Biology 2014, 34 (5), 3437. 10. Kampf, J. P.; Kleinfeld, A. M., An Unknown Protein Mediates Free Fatty Acid Transport Across the Adipocyte Plasma Membrane. Physiology 2007, 22, 7-29. 11. Cupp, D.; Kampf, J. P.; Kleinfeld, A. M., Fatty Acid-Albumin Complexes and the Determination of the Transport of Long Chain Free Fatty Acids across Membranes. Biochemistry 2004, 43 (15), 4473-4481. 12. Kleinfeld, A. M., Lipid phase fatty acid flip-flop, is it fast enough for cellular transport? J Membr Biol 2000, 175 (79-86). 13. Spector, A. A.; John, K.; Fletcher, J. E., Binding of long-chain fatty acids to bovine serum albumin. Journal of lipid research 1969, 10 (1), 56-67. 14. Simard, J. R.; Pillai, B. K.; Hamilton, J. A., Fatty acid flip-flop in a model membrane is faster than desorption into the aqueous phase. Biochemistry 2008, 47 (35), 9081-9089. 15. Kampf, J. P.; Cupp, D.; Kleinfeld, A. M., Different Mechanisms of Free Fatty Acid Flip-Flop and Dissociation Revealed by Temperature and Molecular Species Dependence of Transport across Lipid Vesicles. J Biol Chem 2006, 281, 21566-21574. 16. Kamp, F.; Hamilton, J. A., Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. Biochemistry 1993, 32, 11074-11085. 17. Pillai, B. K.; Jasuja, R.; Simard, J. R.; Hamilton, J. A., Fast diffusion of very long chain saturated fatty acids across a bilayer membrane and their rapid extraction by cyclodextrins: Implications for adrenoleukodystrophy. Journal of Biological Chemistry 2009, 284 (48), 33296-33304. 18. Zhang, F.; Kamp, F.; Hamilton, J. A., Dissociation of long and very long chain fatty acids from phospholipid bilayers. Biochemistry 1996, 35 (50), 16055-16060. 27 ACS Paragon Plus Environment


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28 19. Kamp, F.; Zakim, D.; Zhang, F.; Noy, N.; Hamilton, J. A., Fatty Acid Flip-Flop in Phospholipid Bilayers Is Extremely Fast. Biochemistry 1995, 34 (37), 11928-11937. 20. Liu, Y.; Yan, E. C. Y.; Zhao, X.; Eisenthal, K. B., Surface potential of charged liposomes determined by second harmonic generation. Langmuir 2001, 17 (7), 20632066. 21. Shang, X.; Liu, Y.; Yan, E.; Eisenthal, K. B., Effects of Counterions on Molecular Transport Across Liposome Bilayer: Probed by Second Harmonic Generation. Journal of Physical Chemistry B 2001, 105 (51), 12816-12822. 22. Liu, J.; Subir, M.; Nguyen, K.; Eisenthal, K. B., Second Harmonic Studies of Ions Crossing Liposome Membranes in Real Time. Journal of Physical Chemistry B 2008, 112 (48), 15263-15266. 23. Zeng, J.; Eckenrode, H. M.; Dounce, S. M.; Dai, H.-L., Time-Resolved Molecular Transport across Living Cell Membranes. Biophysical Journal 2013, 104 (1), 139-145. 24. Liu, Y.; Yan, E. C. Y.; Eisenthal, K. B., Effects of bilayer surface charge density on molecular adsorption and transport across liposome bilayers. Biophysical Journal 2001, 80 (2), 1004-1012. 25. Salafsky, J. S.; Eisenthal, K. B., Second harmonic spectroscopy: detection and orientation of molecules at a biomembrane interface. Chemical Physics Letters 2000, 319 (5,6), 435-439. 26. Sharifian Gh, M.; Wilhelm, M. J.; Dai, H. L., Label-Free Optical Method for Quantifying Molecular Transport Across Cellular Membranes in Vitro. Journal of Physical Chemistry Letters 2016, 7 (17), 3406-3411. 27. Lambert, A. G.; Davies, P. B.; Neivandt, D. J., Implementing the Theory of Sum Frequency Generation Vibrational Spectroscopy: A Tutorial Review. 2007. 28. Anglin, T. C.; Cooper, M.; Li, H.; Chandler, K.; Conboy, J. C., Free energy and entropy of activation for phospholipid flip-flop in planar supported lipid bilayers. Journal of Physical Chemistry B 2010, 114 (5), 1903-1914. 29. Brown, K. L.; Conboy, J. C., Electrostatic Induction of Lipid Asymmetry. J. Am. Chem. Soc 2011, 133, 8794-8797. 30. Brown, K. L.; Conboy, J. C., Phosphatidylglycerol Flip-Flop Suppression due to Headgroup Charge Repulsion. 2015. 31. Anglin, T. C.; Brown, K. L.; Conboy, J. C., Phospholipid Flip-flop Modulated by Transmembrane Peptides WALP and Melittin. J Struct Biol 2009, 168 (1), 37-52. 32. Brown, K. L.; Conboy, J. C., Lipid Flip-Flop in Binary Membranes Composed of Phosphatidylserine and Phosphatidylcholine. Journal of Physical Chemistry B 2013, 117 (48), 15041-15050. 33. Allhusen, J. S.; Kimball, D. R.; Conboy, J. C., Structural Origins of Cholesterol Accelerated Lipid Flip-Flop Studied by Sum-Frequency Vibrational Spectroscopy. J. Phys. Chem. B 2016, 120, 3157-3168. 34. Allhusen, J. S.; Conboy, J. C., The Ins and Outs of Lipid Flip-Flop. Accounts of Chemical Research 2017, 50, 58-65. 35. Anglin, T. C.; Conboy, J. C., Lateral pressure dependence of the phospholipid transmembrane diffusion rate in planar-supported lipid bilayers. Biophysical Journal 2008, 95 (1), 186-193.


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29 36. Anglin, T. C.; Conboy, J. C., Kinetics and Thermodynamics of Flip-Flop in Binary Phospholipid Membranes Measured by Sum-Frequency Vibrational Spectroscopy †. Biochemistry 2009, 48, 10220-10234. 37. Liu, J.; Conboy, J. C., Structure of a gel phase lipid bilayer prepared by the Langmuir-Blodgett/ Langmuir-Schaefer method characterized by sum-frequency vibrational spectroscopy. Langmuir 2005, 21 (20), 9091-9097. 38. Marsh, D., Lateral Pressure in Membranes. Biochim. Biophys. Acta. Biomembr. 1996, 1286, 183-223. 39. Plant, A. L., Supported hybrid bilayer membranes as rugged cell membrane mimics. Langmuir 1999, 15 (15), 5128-5135. 40. Liu, J.; Conboy, J. C., Asymmetric Distribution of Lipids in a Phase Segregated Phospholipid Bilayer Observed by Sum-Frequency Vibrational Spectroscopy. Journal of Physical Chemistry C 2007, 111 (25), 8988-8999. 41. Smaby, J. M.; Kulkarni, V. S.; Momsen, M.; Brown, R. E. The Interfacial Elastic Packing Interactions of Galactosylceramides, Sphingomyelins, and Phosphatidylcholines; 1996; pp 868-877. 42. George, L.; Gaines, G., Insoluble Monolayers at Liquid-Gas Interfaces. 1996; p 386-386. 43. Ma, G.; Allen, H. C., Condensing effect of palmitic acid on DPPC in mixed Langmuir monolayers. Langmuir 2007, 23 (2), 589-597. 44. Hac-Wydro, K.; Wydro, P., The influence of fatty acids on model cholesterol/phospholipid membranes. Chemistry and Physics of Lipids2 2007, 150 (1), 66-81. 45. Risoví, D.; Penezí, A.; Vidaˇvidačadě, V.; Suzanašegota, S. S.; Blaženka, B.; Gašparoví, G., Surface free energy tuning of supported mixed lipid layers †. 2016. 46. Rabinovitch, W.; Robertson, R. F.; Mason, S. G., Relaxation of Surface Pressure and Collapse of Unimolecular FIlms of Stearic Acid. Canadian Journal of Chemistry 1960, 38 (2031), 1881-1890. 47. Hac-Wydro, K.; Jedrzejek, K.; Dynarowicz-Latka, P., Effect of saturation degree on the interactions between fatty acids and phosphatidylcholines in binary and ternary Langmuir monolayers. Colloids and Surfaces B: Biointerfaces 2009, 72, 101-111. 48. Lozano, M. M.; Longo, M. L., Complex formation and other phase transformations mapped in saturated phosphatidylcholine/DSPE-PEG2000 monolayers. Soft Matter 2009, 5, 1822-1834. 49. Gong, K.; Feng, S.-S.; Lin Go, M.; Soew, P. H. Effects of pH on the stability and compressibility of DPPC/cholesterol monolayers at the air-water interface; 2002; pp 113-125. 50. Pazin, W. M.; Ruiz, G. C. M.; Oliveira, O. N. d.; Constantino, C. J. L., Interaction of Artepillin C with model membranes: Effects of pH and ionic strength. Biochimica et Biophysica Acta - Biomembranes 2019, 1861 (2), 410-417. 51. Pilch, P. F.; Hamilton, J. A.; Kamp, F.; Guo, W.; Souto, R.; Corkey, B. E., Rapid Flip-flop of Oleic Acid across the Plasma Membrane of Adipocytes. Journal of Biological Chemistry 2003, 278 (10), 7988-7995. 52. Leekumjorn, S.; Sum, A. K., Molecular Characterization of Gel and LiquidCrystalline Structures of Fully Hydrated POPC and POPE Bilayers. J Phys Chem B 2007, 111 (21), 6026-6033. 29 ACS Paragon Plus Environment


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30 53. Lee, C.; Bain, C. D., Raman spectra of planar supported lipid bilayers. Biochimica et Biophysica Acta 2005, (1711), 59-71. 54. Kamp, F.; Hamilton, J. A., pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids. Proc Natl Acad Sci 1992, 89, 11367-11370. 55. Wei, C.; Pohorille, A., Flip-flop of oleic acid in a phospholipid membrane: rate and mechanism. J Phys Chem B 2014, 118 (45), 12919-12926. 56. Armstrong, V. T.; Brzustowicz, M. R.; Wassall, S. R.; Jenski, L. J.; Stillwell, W., Rapid flip-flop in polyunsaturated (docosahexaenoate) phospholipid membranes. Archives of Biochemistry and Biophysics 2003, 414 (1), 74-82. 57. Liu, J.; Conboy, J. C., 1,2-Diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy. Biophysical Journal 2005, 89 (4), 25222532. 58. 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 Discussions 2013, 161, 45-61. 59. Mercado, F. V.; Maggio, B.; Wilke, N., Phase diagram of mixed monolayers of stearic acid and dimyristoylphosphatidylcholine. Effect of the acid ionization. Chemistry and Physics of Lipids 2011, 164 (5), 386-392. 60. Wydro, P.; Hac-Wydro, K., Thermodynamic description of the interactions between lipids in ternary Langmuir monolayers: the study of cholesterol distribution in membranes. Journal of Physical Chemistry B 2007, 111 (10), 2495-2502. 61. Dynarowicz-Łatka, P.; Hac-Wydro, K., Interactions between Phosphatidylcholines and Cholesterol in Monolayers at the Air/Water Interface. Colloids Surfaces B: Biointerfaces 2004, 37, 21-25. 62. Krug, R. R. H., W. G.; Grieger, R. A. J., Enthalpy-entropy compensation. 1. Some fundamental statistical problems associated with the analysis of van't Hoff and Arrhenius data. 80 1976, 2335-2341 (21). 63. Krug, R. R. H., W. G.; Grieger, R. A. J., Statistical interpretation of enthalpy– entropy compensation. 261 1976, 566-56. 64. Lumry, R. R., S. , Enthalpy–entropy compensation phenomena in water solutions of proteins and small molecules: A ubiquitous properly of water. Biopolymers 1970, 9 (10), 1125-1227. 65. Gilli, P. F., V.; Gilli, G.; Borea, P.A., Enthalpy-entropy compensation in drugreceptor binding. J. Phys. Chem. 1994, 98 (5), 1515-1518. 66. Thomas, R. M.; Baici, A.; Werder, M.; Schulthess, G.; Hauser, H., Kinetics and Mechanism of Long-Chain Fatty Acid Transport into Phosphatidylcholine Vesicles from Various Donor Systems. Biochemistry 2002, 41, 1591−1601-1591−1601.


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31 Figures

Figure 1. Proposed model for fatty acid transport, adapted from reference 10. A fatty acid (purple) dissociates from a serum albumin (gray), followed by adsorption onto the plasma membrane at a rate of kads. Afterwards, the fatty acid flips to the cytosolic side of the plasma membrane at a rate kff. In the final step, the FA desorbs from the plasma membrane and into the cytosol at a rate kdes.



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32

Figure 2. From left to right, pressure-area isotherms of pure DSPC (black), 1:7 SA:DSPC (blue), 1:3 SA:DSPC (green), 1:1 SA:DSPC (red), and pure SA (gray) on a subphase of 150 mM PBS at a of pH 7.4 recorded at 22 ± 1 °C.


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33

Figure 3. Mean molecular area of binary SA:DSPC monolayers measured at 30 mN/m from the pressure-area isotherms in Figure 1, as a function of mol% SA. The dashed line denotes the predicted mean molecular area for an ideal mixture, calculated using eq. 5.

Figure 4. Free energy of mixing (Gexc) as a function of mol% SA, calculated at 30 mN/m.



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34

Figure 5. Compression moduli calculated for pure DSPC (black), 1:7 SA:DSPC (blue), 1:3 SA:DSPC (green), 1:1 SA:DSPC (red), and pure SA (gray).


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35

4

4

A

B

3

SFVS Intensity (a.u.)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2

1

0 2750

3

2

1

0

2800

2850

2900

2950

3000

Wavenumbers (cm-1)

3050 2750

2800

2850

2900

2950

-1

3000

3050

Wavenumbers (cm )

Figure 6. (A) SFVS spectra of the DSPC component for SAd3:DSPC bilayers with the following compositions: 0:1 SAd3:DSPC (black), 1:7 SAd3:DSPC (dark blue), 1:3 SAd3:DSPC (dark green), and 1:1 SAd3:DSPC (dark red). Peak assignments are shown in gray: CH2 s (2848 cm-1, dashed), CH3 s (2875 cm-1, solid), CH2 as (2898 cm-1, dots), CH3 FR (2936 cm-1, dots), and CH3 as (2952 cm-1, dots). (B) SFVS spectra of the SA component for SA:DSPCd70 bilayers with the following compositions: 1:0 SA:DSPCd70 (gray), 1:7 SA:DSPCd70 (blue), 1:3 SA:DSPCd70 (green), and 1:1 SA:DSPCd70 (red). Peak assignments are shown in gray: CH3 s (2875 cm-1, solid), CH3 FR (2936 cm-1, dots), and CH3 as (2952 cm-1, dots). All spectra were recorded using PBS in D2O, have been normalized to the CH3 S stretch and offset for clarity.



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36

Figure 7. Representative CH3 vs intensity decays for the SA component of a pure SA bilayer (gray), 1:7 SA:DSPCd70 (12.5 mol%, light blue), 1:3 SA:DSPCd70 (25 mol%, green), and 1:1 SA:DSPCd70 (50%, red). The solid lines are the fits to data using eq. 4.


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37

Figure 8. CH3 S intensity decay curves for the proteated SA component present in either the proximal (top, blue) or distal (bottom, orange) leaflet recorded at 20˚C in PBS (D2O). The solid lines correspond to the fits of the decays using eq. 4. The calculated kff of SA in the proximal and distal leaflets were (8.35 ± 0.08)×10-5 s-1 and (8.19± 0.07)×10-5 s-1, respectively. The decays were normalized to the initial CH3 νS intensity and offset for clarity.



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38

Figure 9. Comparison of SA and DSPC flip-flop kinetics in the SA:DSPC bilayers over a range of temperatures. The rates of SA flip-flop in the PSLBs are shown with solid circles: pure SA (gray, 100 mol%), 1:7 (blue, 12.5 mol%), 1:3 (green, 25 mol%), and 1:1 (red, 50 mol%). The rates of DSPC flip-flop shown with open circles: pure DSPC (black, 100 mol%), 1:7 (navy, 87.5 mol%), 1:3 (dark green, 75 mol%), and 1:1 (dark red, 50 mol%). Errors bars for the rates were obtained from the nonlinear least-squares regression of the CH3 vs intensity decays.


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39

Figure 10. Representative CH3 S intensity decays for the DSPC component of a pure DSPC bilayer (gray), 1:7 SAd3:DSPC (87.5 mol%, blue), 1:3 SAd3:DSPC (75 mol%, green), and 1:1 SAd3:DSPC (50%, red). All decays were recorded at 46˚C in PBS (D2O). The solid lines are the fits to the data using eq. 4.



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40

Figure 11. Gibbs plot of the free energy of activation as a function of temperature for SA and DSPC. The lines correspond to the fits of each data set to eq. 11.


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41

Figure 12. Activation enthalpy-entropy compensation for SA (filled circles, for SA:DSPCd70 bilayers with the following compositions: 1:0 SA:DSPCd70 (gray), 1:7 SA:DSPCd70 (blue), 1:3 SA:DSPCd70 (green), and 1:1 SA:DSPCd70 (red)) and DSPC (open circles, for the following compositions: 0:1 SAd3:DSPC (black), 1:7 SAd3:DSPC (dark blue), 1:3 SAd3:DSPC (dark green), and 1:1 SAd3:DSPC (dark red)).The solid line is a fit to the data using eq. 12.



42

2.0

1.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 45

1.0

0.5

0.0 1.5 1.0 0.5 0.0 2750 2800 2850 2900 2950 3000 3050

Wavenumbers (cm-1) Figure 14. (A) SFVS spectrum of a 1:1 SA:DSPCd70 monolayer on a MTES coated and (B) SFVS spectrum of the SiO2/MTES surface. All spectra were recorded at 23 °C in PBS (D2O).


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43

Figure 15. Intensity of the CH3 S of SA for HSLBs composed of 1:1 (red, line), 1:3 (green, line), and 1:7 SA:DSPCd70 (blue, line) as a function of time. All curves were recorded at 20 ˚C in PBS (D2O). The data were normalized to the initial CH3 S intensity and offset for clarity. The dips at 1.3 hours indicate when the incident beams were blocked, while the flow cell was flushed with PBS (D2O).


44


Table 1. The measured flip-flop rates and corresponding half-lives for SA and DSPC as a function of concentration and temperature. Errors in the rates correspond to errors in the fits to the experimental CH3 vs intensity decays with time. Errors in the half-life are propagated from the errors in the rates. Mol% SA 12.5 %

T (˚C)

k (s-1)  105

t1/2 (min)

17.6 ± 0.1 18.0 ± 0.1 19.6 ± 0.3 26.8 ± 0.5

5.6 ± 0.5 7.6 ± 0.2 10.8 ± 0.5 31.6 ± 0.2

102 ± 8 75 ± 1 53 ± 2 18.3 ± 0.1

25%

15.5 ± 0.1 16.5 ± 0.2 19.2 ± 0.2 26.4 ± 0.1 18.1 ± 0.1 19.8 ± 0.4 21.0 ± 0.4 25.6 ± 0.4 27.4 ± 0.3 31.0 ± 0.5 32 ± 1 16.8 ± 0.4 20.30 ± 0.03 22.49 ± 0.04 25.6 ± 0.6

6.02 ± 0.02 8.58 ± 0.03 27.2 ± 0.4 126.7 ± 0.8 4.23 ± 0.04 6.93 ± 0.08 9.6 ± 0.2 17.2 ± 0.6 30.8 ± 0.5 62 ± 2 74 ± 2 5.8 ± 0.2 20.2 ± 0.4 22.4 ± 0.5 41 ± 2

96.0 ± 0.3 67.3 ± 0.3 21.2 ± 0.2 4.6 ± 0.3 136.6 ± 1 83.4 ± 0.9 60 ± 1 33.6 ± 0.6 18.8 ± 0.3 9.3 ± 0.3 7.8 ±0 .2 100 ± 4 28.6 ±0.5 25.8 ± 0.6 14.2 ± 0.8

50%

100%

Mol% DSPC 87.5%

75%

50%

100%

T (˚C)

k (s-1)  105

t1/2 (min)

45.1 ± 0.1 45.7 ± 0.3 49.0 ± 0.1 50.1 ± 0.1 52.5 ± 0.1 39.6 ± 0.1 42.3 ± 0.1 43.6 ± 0.1 45.90 ± 0.06 44.4 ± 0.3 46.6 ± 0.1 49.1 ± 0.1 52.8 ± 0.1 52.90 ± 0.04

0.425 ± 0.005 0.730 ± 0.003 1.30 ± 0.01 1.778 ± 0.005 3.270 ± 0.009 0.91 ± 0.02 1.25 ± 0.07 1.322 ± 0.002 2.59 ± 0.02 0.860 ± 0.002 1.215 ± 0.005 1.445 ± 0.007 1.95 ± 0.02 2.03 ± 0.01

1360 ± 10 791 ±3 443 ±3 324.9 ±0.7 176.7 ±0.5 634.0 ± 10 462 ± 2 436.8 ± 0.6 223 ± 2 670 ± 10 475 ± 2 400 ± 2 296 ± 3 285 ± 1

46.2 ± 0.1

6.62 ± 0.01

86.0 ± 0.1

Table 2. Transition state free energy for SA and DSPC. The free energies of SA flip-flop were calculated at 295 K, while the free energies of DSPC flip-flop were calculated at 319 K. SA Flip-Flop ΔG‡ Mol% DSPC DSCP Flip-Flop ΔG‡ Mol% SA (kJ/mol) (kJ/mol) 100% 102 ± 20 12.5% 94 ± 15 87.5% 105 ± 3 25% 91 ± 3 75% 106 ± 2 50% 95 ± 6 50% 106 ± 3 100% 92 ± 40


44

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TOC


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Determination of the Rate-Limiting Step in Fatty Acid Transport | The

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