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The Transport and Organization of Cholesterol in Planar SolidSupported Lipid Bilayer Depend on the Phospholipid Flip-Flop Rate Ting Yu, Guangnan Zhou, Xia Hu, and Shuji Ye Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02560 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016
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The Transport and Organization of Cholesterol in Planar Solid-Supported Lipid Bilayer Depend on the Phospholipid Flip-Flop Rate Ting Yu,1,2 Guangnan Zhou,1 Xia Hu,1,2 Shuji Ye,1,2,*
1
Hefei National Laboratory for Physical Sciences at the Microscale, and Department of
Chemical Physics, and 2Synergetic Innovation Center of Quantum Information & Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026, China
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Abstract Understanding the transport behavior of the cholesterol molecules within cell membrane is a key challenge in cell biology at present. Here, we applied sum frequency generation vibrational spectroscopy (SFG-VS) to characterize the transport and organization of the cholesterol in different kinds of planar solid-supported lipid bilayers by combining achiral and chiral-sensitive polarization measurements. This method allows us to distinguish cholesterol organizations in the tail to tail, head to tail, head to head, and side-by-side manners. It is found that the movement of cholesterol in the lipid bilayer largely depends on the flip-flop rate of the phospholipid. The flip-flop dynamics of the phospholipid and cholesterol is synchronous. In solid-supported zwitterionic phosphocholine lipid bilayer, the cholesterol molecules flip quickly from the distal leaflet to the neutral proximal leaflet of the bilayer and form tail to tail organization on both leaflets. The phosphocholine lipid and cholesterol show the same flip-flop rate. However, when the proximal leaflet is prepared using negative glycerol phospholipids, cholesterol organizes itself by mainly forming α-β structure on the distal leaflet. Due to the strong interaction between glycerol phospholipid and substrate, no or only partial cholesterol molecules flip from the distal leaflet to the negatively charged proximal leaflet. But the cholesterol molecules undergo flip-flop in the presence of salt solution because the ions weaken the interaction between the negative phospholipid and the substrate.
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1. Introduction The cellular cholesterol homeostasis between membranes and within a membrane plays very important roles in human health and normal cellular functions over the lifespan of the cell.1-3 The abnormal abundance and distribution of cholesterol within cells can lead to many diseases such as Alzheimer’s disease, arteriosclerosis or other cardiovascular diseases.4-6 The transverse motion of cholesterol between bilayer leaflets (also known as flip-flop) is essential to regulate the total cellular level and distribution of cholesterol within cells and may contribute to the formation of lipid raft.1-3,7-9 Because of this, extensive researches have been performed to investigate the cellular cholesterol trafficking using various experimental and theoretical methods.7-15 Despite many progresses, the factors that control the cholesterol transport dynamics are still obscure. Many scientific problems associated with cellular cholesterol trafficking and compartmentalization remain elusive so far.7 In addition, earlier reports indicated that the flip-flop rates and pathways depend on the experimental methods.9 The studies by fluorescence spectroscopy,10 electron spin resonance,11 and molecular dynamic simulations8, 9 suggested that cholesterol can flip within a few seconds or even milliseconds.12 In contrast, the studies approached by time-resolved small-angle neutron scattering technique revealed that the flip-flop of cholesterol in the bilayer has a half-time of 200 min at 50 °C.14 Because most of the previous studies require the introduction of fluorescence or spin labels into the lipids, which may change the physical and chemical properties of membrane and thus affect the flip-flop behavior of cholesterol,16,17 it is apparently critical to explore label-free tool to identify the motion of the cholesterol in the cell membrane and elucidate how the experimental parameters influence the cholesterol transport dynamics.
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Sum frequency generation vibrational spectroscopy (SFG-VS) is a second-order optical laser technique which permits to identify the molecular species (or chemical groups) on a surface or at an interface in situ in real time without exogenous labels.18-20 SFG-VS has been applied to study the structure and orientation of various biomolecules including cholesterol in the interfacial environments.21-30 The SFG signal is proportional to the square of the effective ( 2) of the vibrational mode and the intensity of the second-order nonlinear susceptibility χ eff
two input beams I1 (ωvis ) and I 2 (ω IR ) (Eq.(1)), which vanishes for the material having inversion symmetry under the dipole approximation.18-20 2
( 2) I (ω SFG ) ∝ χ eff I1 (ωvis ) I 2 (ω IR )
(1)
where ωSFG, ωvis, and ωIR are the frequency of the sum-frequency beam, visible and infrared input beams, respectively. Therefore, the signals generated from the chemical groups that symmetrically arrange in both leaflets of the bilayer can effectively cancel each other. Because of these symmetry constraints, SFG-VS has been demonstrated to be a robust and label-free tool to investigate the flip-flop behavior of phospholipid bilayer and measure the rate of transbilayer movement by probing the decay in the CH3 (or CD3) symmetric stretching intensity with time.17, 31-34 However, most of previous studies in this area mainly focus on the cholesterol condensing effect on phospholipid monolayer,26-28 or the flip-flop behavior of phospholipid itself,17, 31-35 rather than the transport of cholesterol. Recently, we successfully exploited SFG-VS to track assembly behavior of the cholesterol analogue 6-ketocholestanol (6-KC) in the planar solid-supported lipid bilayer.36 It is achieved by monitoring the methyl groups in the isooctyl hydrocarbon tail of 6-KC and the methyl substituents of sterol ring using achiral-sensitive ssp (s-polarized SFG output, s-polarized visible input, p-polarized
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infrared input) and chiral-sensitive psp polarization measurements, respectively. Here, because the three-dimensional characteristics of chirality require the three subindices ijk of ) to be all different (i ≠ j≠ k), the polarization the second-order nonlinear susceptibility χ R( 2,ijk
combinations such as ssp, ppp, and sps are termed as achiral polarizations while the combinations of psp, spp, and pps are chiral polarizations and sensitive to the molecular chirality.18-20 Such a combination of achiral and chiral polarization measurement offers a powerful tool in directly distinguishing the possible forms in the organizations of cholesterol in lipid bilayer (Figure S1). Cholesterol is a molecule comprising a flexible isooctyl hydrocarbon tail and a near planar steroid ring (denoted as the head of cholesterol) (Figure 1). The methyl group in the isooctyl hydrocarbon tail is achiral. The plane of the steroid ring is asymmetric. We refer the flat and smooth side without substituents as the α face and the rough side with substituents of chiral methyl groups as β face (Figure 1C). In terms of the symmetry constraints on SFG, different organization in Figure S1 will lead to different SFG response of the methyl groups in isooctyl hydrocarbon tail and sterol ring. When cholesterol flips from one leaflet to another leaflet and organizes itself in tail to tail manners (Figure S1d, S1e, and S1f), the achiral ssp or ppp signals from the methyl groups in the isooctyl hydrocarbon tail can be effectively canceled. By contrast, when cholesterol organizes in the manners of Figure S1a, S1b and S1c (the cholesterol is only distributed in one leaflet of the bilayer), the ssp or ppp signals of methyl group in the tail cannot be cancelled. Accordingly, the chiral psp signals from the methyl substituents of sterol ring can be effectively eliminated with the formation of
α-α and β-β structures (Figure S1b, S1c, and S1d) because the methyl substituents in the cholesterol pair orient in the opposite direction. Otherwise, formation of α-β structures whose
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methyl substituents in the cholesterol pair orient in the same direction could produce large chiral psp signals (Figure S1a, S1e and S1f).36 Here, the side-by-side organization of the α-α,
β-β and α-β structures is α face by α face, β face by β face, and α face by β face, respectively. The detailed discussion of this method has been described in our recent publication.36 We won’t repeat here to avoid the overlap. We can anticipate that this method can be used to elucidate the dynamics of cholesterol flipping and assembling in a cell environment. Herein, we applied this new method to investigate the influence of phospholipid types and the presence of salt solution on the cholesterol flipping and organization in lipid bilayer to elucidate how the environmental conditions govern the cholesterol flip-flop dynamics.
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Figure 1. The molecular structures of cholesterol and the near total internal reflection experimental geometries used for SFG collection. A) Cholesterol; B) Cholesterol-d7; C) The α face (the smooth and flat side) and β face (the rough side with chiral methyl groups) of cholesterol; D) A schematic in the xz plane for the phospholipids monolayer deposited at the surface of CaF2 prism; E) Three dimensional schematic for the phospholipids monolayer in Figure 1D; F) A schematic in the xz plane for the bilayer which was prepared by contacting the CaF2 prism’s monolayer-coated right-angle face with the monolayer of the mixture of phospholipids and cholesterol (or cholesterol-d7) at the water surface; G) Three dimensional schematic for the proximal and distal leaflets in Figure 1F.
2. Experimental Section 2.1 Materials and Sample Preparations Cholesterol (Chol, Figure 1A) and cholesterol-d7 (d-Chol, Figure 1B) were purchased from
Sigma-Aldrich
with
a
purity
of
1,2-dimyristoyl-sn-glycerol-3-phosphocholine phosphocholine
(DPPC),
≥
98%.
(DMPC),
1,2-distearoyl-sn-glycero-3-
The
phospholipids
of
1,2-dipalmitoyl-sn-glycero-3phosphocholine
(DSPC),
1,2-dimyristoyl-d54-sn-glycero-3- phosphocholine- 1,1,2,2-d4-N,N,N-trimethyl-d9 (d-DMPC), 1,2-dipalmitoyl-d62-sn-glycero-3- phosphocholine-1,1,2,2-d4-N,N,N-trimethyl-d9 (d-DPPC), 7
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1,2-distearoyl-d70-sn-glycero-3 1,2-dimyristoyl-sn-
glycero-3-
1,2-dipalmitoyl-sn-glycero-3-
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-phosphocholine-1,1,2,2-d4-N,N,N-trimethyl-d9 phospho-(1'-rac-glycerol) phospho-(1'-rac-glycerol)
(sodium (sodium
(d-DSPC),
salt)
(DMPG),
salt)
(DPPG),
1,2-distearoyl-sn-glycero-3- phospho-(1'-rac-glycerol) (sodium salt) (DSPG), 1,2-dimyristoyl -d54-sn-glycero-3- [phospho-rac-(1-glycerol)] (sodium salt) (d-DMPG), 1,2-dipalmitoyl -d62-sn-glycero-3 -[phospho-rac-(1-glycerol)] (sodium salt) (d-DPPG), 1,2-distearoyl-d70-snglycero-3- [phospho-rac-(1-glycerol)] (sodium salt) (d-DSPG) were purchased from Avanti Polar Lipids (Alabaster, AL). Chol and d-Chol were dissolved in methanol (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a concentration of 1.0 mg/mL and stored at -20 °C. DXPC and d-DXPC(X=M, P, or S) were dissolved in chloroform (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a concentration of 1.0 mg/mL. DXPG and d-DXPG(X=M, P, or S) were dissolved in chloroform and methanol (with a volume ratio of 65:35) at a concentration of 1.0 mg/mL. All of phospholipids were stored at -20 °C. The molecular structures of the phospholipids are given in Figure S2. The potassium chloride (KCl) was purchased from Sinopharm Chemical Reagent Co., Ltd. with a purity of > 99.5%. The salt was baked at around 500 °C for more than 8 h in order to remove the organic impurities. The potassium chloride (KCl) was dissolved into ultrapure water from a Milli-Q reference system (Millipore, Bedford, MA) with a concentration of 0.05 mol/L. Right angle prisms (CaF2) were purchased from Chengdu Ya Si Optoelectronics Co., Ltd (Cheng Du, China). The CaF2 prism was chosen as the substrate because it is weakly positively charged at neutral pH conditions (surface potential approximately +35 mV at pH 7.5)37 and insoluble in water. Furthermore, it is transparent in a wider wave number range, particularly in the frequency of 1950-2300 cm-1,
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while most of common substrates (for example, silica) is almost not transparent in the frequency of 1950-2300 cm-1 and not suitable to monitor the spectra from the CD2 and CD3 vibrational modes. The prism cleaning and lipid bilayer preparation were carried out using a standard procedure which has been given in our previous publications.36 The experimental geometries are described in Figure 1D and 1F. Here we first deposited the phospholipid monolayer on CaF2 prism surface to produce the monolayer/air surface using Langmuir-Blodgett method at a KSV mini trough LB system at the surface pressure of 35 mN/m (Figure 1D and 1E). The bilayer was prepared in situ using Langmuir-Schaefer method by contacting the CaF2 prism’s monolayer-coated right-angle face with the monolayer of the mixture of phospholipid and Chol (or d-Chol) with the surface pressure of 35 mN/m at the water surface (Figure 1F and 1G). The molecular ratio of the mixture of phospholipid and Chol (or d-Chol) is 1:1. The mixture was not deposited on the prism surface. Brewster angle microscopy (BAM) experiments indicate that the mixture of phospholipid and cholesterol (or cholesterol-d7) do not phase-segregation at this ratio. For this molecular ratio, the mixture is in the liquid-ordered phase at 24°C.38, 39 In addition, at 24 °C, pure phospholipid monolayer of DMPC and DMPG are in fluid phase while DPPC，DSPC，DPPG and DSPG are in gel phase. We referred the leaflet next to the solid support to the proximal leaflet and the leaflet far from the solid support to the distal leaflet (Figure 1F).
2.2 SFG-VS Experiments The theories and instruments of SFG-VS have been introduced previously.18-20 The SFG-VS experimental setup has been described in our earlier reports.40 Our SFG system is
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put in the ultra-clean laboratory with constant temperature and humidity. All the experiments were carried out at room temperature (24℃). The energy of visible beam and IR beam at 2000 -2300cm-1 and 2800-3000 cm-1 is about 10 µJ and 40~50µJ, respectively. Such laser power does not cause the damage of the phospholipid, which is confirmed by measuring the SFG intensity of the phospholipid monolayer at the CaF2 prism surface. The intensity does not change after longtime continuous irradiation. Actually, previous SFG studies on the phospholipid already verified that.24-29,31,34,35 The SFG-VS signals from cholesterol and phospholipid molecules were collected with different polarization combination, including ssp, ppp and psp. Each of SFG-VS spectral data point was averaged over 100 times and normalized by the energy of input IR and visible beams.
3. Results and Discussion 3.1 The Transport of Cholesterol in Neutral PC Phospholipid Bilayer As a powerful tool to monitor the formation or the loss of asymmetry of lipid bilayer, SFG has been applied to investigate the effect of cholesterol and peptides on the flip-flop of lipid bilayer.25 It was suggested the peptides can promote the flip-flop rate of phospholipid in neutral lipid bilayer.31 Generally, the SFG intensity is related to the Fresnel coefficients, the effective surface molecule number (∆Ns), and the average orientation angle (θ)(see Eqs.(1)-(3)).18-20, 36 ( 2) ssp ( 2) χ eff( 2), ssp = L yy (ω SFG ) L yy (ωvis ) Lzz (ω IR ) sin θ IR χ yyz = C Fresnel χ yyz
(2)
( 2) χ yyz = ∆N s d yyz [< cosθ > −c yyz < cos 3 θ >]
(3)
where Lii(ω) is the diagonal elements of Fresnel factor at frequency ω. θIR is the incidence angle of the IR beam. θ is the orientation angle of a chemical group. The effective surface
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molecule number (∆Ns) is referred to the molecules that have a net contribution to the SFG ( 2) is the element tensor of the second-order susceptibility in the lab coordinates. In signal. χ yyz
theory, a line shape such as peak center and linewidth of a vibrational mode is sensitive to a molecule’s environment or the phase state. Therefore the change of line shape may affect the intensity of a single wavelength. However, it has been evident that the flip-flop process mainly causes the change of the effective surface molecule number (∆Ns), rather than the line shape and the orientation angle of CD3.17,31-34 Therefore, the flip-flop rate of d-Chol can be determined by continuously measuring SFG intensity of peak center from the CD3 group of the cholesterol tail as a function of time. Such method has been applied to measure the flip-flop rate of phospholipids.17,31-34 The SFG spectra of the CD3 group are dominated by a strong peak at ~2075 cm-1, which is attributed to CD3 symmetric stretch.41,42 Here, we first considered the transport of cholesterol in three neutral PC phospholipid bilayers: DMPC/(DMPC+d-Chol), DPPC/(DMPC+d-Chol), and DSPC/(DMPC+d-Chol). We denoted these bilayers as DXPC/(DMPC+d-Chol), X=M, P, or S. We used the cholesterol molecules with an isotopically methyl groups in the isooctyl hydrocarbon tail to distinguish SFG signal of the cholesterol from the phospholipid bilayer. Figure 2A shows the time-dependent ssp intensity after contacting the DXPC monolayer at CaF2 prism surface with the monolayer of the mixture of DMPC and d-Chol at the water surface. For all of the bilayers DXPC/(DMPC+d-Chol), the ssp signal at 2075 cm-1 increases quickly to a plateau in 20 seconds and becomes stable, which is not influenced by the phospholipid chain length. The intensity change at 2075cm-1 indicates that cholesterol molecules in neutral bilayer show a very fast flip-flop, with flip-flop half-life (t½) of less than 0.2 min at 24°C. After the flip-flop
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reaches equilibrium, we measured the SFG spectra with different polarization combinations. It can be seen that no signals from the d-Chol CD3 groups are observed in the ssp (Figure 2B) and ppp (Figure 2C) spectra. The absence of the signals may come from two contributions: one is because the cholesterol molecules flip quickly from the distal leaflet to the proximal leaflet of the bilayer and form tail to tail organization on both leaflets (Figure S1d-1f); the second is because there are no cholesterol molecules in the bilayer. However, as will be discussed later in this paper, the presence of the chiral signal from the chiral methyl substituents of cholesterol can rule out the second contribution. Considering the absence of the SFG signals from the d-Chol CD3 groups in Figure 2B and 2C, the intensity increases in Figure 2A will originate from the non-resonant background due to the surface change from the air to the water, which can cause several times increase in the Fresnel coefficients.43,44 For ssp in Eq.(2)) at the water our experimental geometry, the Fresnel coefficient of ssp ( C Fresnel
interface is about 4.8 times larger than that at the air surface.44 According to Eq.(S2), the ( 2) effective surface nonlinear susceptibility ( χ eff ) is contributed by the non-resonant ( 2) ( 2) background ( χ NR ) and resonant signals ( χ Re s ) of a vibrational mode. Apparently, when the
flip-flop process takes place very fast (for example, faster than the time resolution of spectra collection) upon formation of DXPC/(DMPC+d-Chol) bilayer, the resonant signals will be very small because the SFG signals from the CD3 groups of d-Chol in the proximal and distal leaflets are canceled effectively. For this case, the total SFG intensity is dominated by the non-resonant background at t>0. In Figure 2A, at t0, the interface is changed from
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the air surface (t 0) and the signals at t>0 come from the bilayer/water interface. Because the non-resonant background in the water interface is larger than that in the air surface, therefore we observed a signal increase in Figure 2A. A
B
a)
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Figure 2. A) The time-dependence of the ssp SFG intensity at 2075 cm-1 during the formation of the lipid bilayer. B) The ssp SFG spectra in the frequency region from 1950-2300 cm-1 after flip-flop reaches equilibrium. C) The ppp SFG spectra in the frequency region from 1950-2300 cm-1 after flip-flop reaches equilibrium. a) DMPC/(DMPC+d-Chol); b) DPPC/(DMPC+d-Chol); c) DSPC/(DMPC+d-Chol). A
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Figure 3. A) The time-dependence of the ssp SFG intensity at 2075 cm-1 during the formation of the lipid bilayer. B) The ssp SFG spectra in the frequency region from 1950-2300 cm-1 after flip-flop reaches equilibrium. C) The ppp SFG spectra in the frequency region from
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1950-2300 cm-1 after flip-flop reaches equilibrium. a) d-DMPC/(DMPC+Chol); b) d-DPPC/(DMPC+Chol); c) d-DSPC/(DMPC+Chol). To understand the dependence of the cholesterol transport dynamics on the phospholipid flip-flop behavior, we further investigated the phospholipid flip-flop dynamics using the bilayer of d-DXPC/(DMPC+Chol). At this case, we prepared the proximal leaflet with deuterated phospholipid. But the cholesterol is not deuterated. Figure 3 shows the time-dependence of the ssp SFG intensity at 2075 cm-1 from the CD3 group of the phospholipid (Figure 3A), and the ssp and ppp SFG spectra after flip-flop reaches equilibrium(Figure 3B and 3C). In Figure 3A, at t
