Letter pubs.acs.org/JPCL
In Situ and Real-Time SFG Measurements Revealing Organization and Transport of Cholesterol Analogue 6‑Ketocholestanol in a Cell Membrane Sulan Ma,†,‡,# Hongchun Li,†,‡,§,# Kangzhen Tian,†,‡,§ Shuji Ye,*,†,‡,§ and Yi Luo†,‡,§,⊥ †
Hefei National Laboratory for Physical Sciences at the Microscale, ‡Department of Chemical Physics, and §Synergetic Innovation Center of Quantum Information & Quantum Physics, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China 230026 ⊥ Department of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology, S-10961 Stockholm, Sweden S Supporting Information *
ABSTRACT: Cholesterol organization and transport within a cell membrane are essential for human health and many cellular functions yet remain elusive so far. Using cholesterol analogue 6-ketocholestanol (6-KC) as a model, we have successfully exploited sum frequency generation vibrational spectroscopy (SFG-VS) to track the organization and transport of cholesterol in a membrane by combining achiral-sensitive ssp (ppp) and chiral-sensitive psp polarization measurements. It is found that 6-KC molecules are aligned at the outer leaflet of the DMPC lipid bilayer with a tilt angle of about 10°. 6-KC organizes itself by forming an α−β structure at low 6-KC concentration and most likely a β−β structure at high 6-KC concentration. Among all proposed models, our results favor the so-called umbrella model with formation of a 6-KC cluster. Moreover, we have found that the long anticipated flip-flop motion of 6-KC in the membrane takes time to occur, at least much longer than previously thought. All of these interesting findings indicate that it is critical to explore in situ, real-time, and label-free methodologies to obtain a precise molecular description of cholesterol’s behavior in membranes. This study represents the first application of SFG to reveal the cholesterol−lipid interaction mechanism at the molecular level. SECTION: Surfaces, Interfaces, Porous Materials, and Catalysis
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Such disputations mainly result from inherent difficulties in labeling and tracking the lipid and cholesterol molecules. Fluorescence spectroscopy and electron spin resonance are the most widely used methods to study intracellular transport processes,18 for which the fluorescence or spin labels have to be introduced into the cholesterol and lipids. It is noticed that exogenous labels may change the intrinsic physical and chemical properties,19 leading to a fast flipping rate.12 In situ, real-time, and label-free methodologies are highly desirable to access a real and complete picture of cholesterol actions. In this study, we have successfully exploited sum frequency generation vibrational spectroscopy (SFG-VS) to probe the organization and transport of cholesterol in membranes in situ by combining achiral- and chiral-sensitive polarization measurements. SFG-VS, a powerful label-free technique for studying molecular structures at the interface,20−22 is a highly surface sensitive method that allows measurement of the transbilayer movement of phospholipids in cellular membranes.23 It has been applied to investigate the cholesterol condensing effect on
holesterol is a major and ubiquitous component of mammalian plasma membranes. It cannot only determine the physiochemical properties of cell membranes but also modulate the interfacial enzyme activities and trigger the raft formation in sphingolipids.1−3 It is well-known that its abundance and distribution within cells are strongly associated with Alzheimer’s disease and other severe neurodegenerative disorders.4 The maintenance of cellular cholesterol homeostasis is therefore critical for human health and normal cellular functions.1−3 Molecular-level understanding of the nature of cholesterol−lipid interactions and cellular cholesterol transport will undoubtedly provide important clues to regulate cholesterol’s behavior within cells in a desired manner.5−7 Currently, several conceptual structural models for the cholesterol−lipid interactions have been proposed,2,6,7 namely, the condensed complex model,3,8 the superlattice model,9 and the umbrella model.10 The motion of the cholesterol in a cell membrane is often considered in a flip-flop manner, but different flip-flop time has been reported ranging from several hours11−13 to a few seconds or even milliseconds,5,6,14−16 depending on the experimental methods.5 Moreover, it has also been suggested that certain flippase is required for the flip-flop in cellular plasma membranes that are rich in cholesterol.17 © 2014 American Chemical Society
Received: November 22, 2013 Accepted: January 7, 2014 Published: January 7, 2014 419
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lipid monolayers.24−28 On the basis of the symmetry constraints and chiral theory of SFG,29 it is known that the SFG signal measured with achiral-sensitive polarization (such as ssp, ppp, and sps) is generated from the macromolecular achiral structure, while the signal measured with chiral-sensitive psp polarization arises solely from the molecules that are arranged in the macromolecular chiral structure.29 The cholesterol molecule comprises a near-planar sterol ring and a flexible achiral isooctyl tail. The sterol ring has a smooth and flat α side without a substituent and a rough β side with chiral methyl substitutions. Consequently, the psp spectra can be used to monitor the structure change of chiral methyl substitutions. In this case, the interferences from the lipid, cholesterol isooctyl tail, and membrane-bound water molecules can be eliminated because they are all silent in psp spectra. Moreover, the cholesterol organizations in the tail (head) to tail (head) and α (β) to α (β) manners produce different symmetry. For example, there are three possible forms of side-by-side organizations on one leaflet, α−α, β−β, and α−β (Figure 1B). It means that the resulting ssp and psp intensity should
a model to investigate the interactions between cholesterol and a neutral lipid bilayer in situ and in real time. 6-KC has a similar structure to cholesterol except with a keto moiety at the position of the B ring (Figure 1A), which can be used to determine the 6-KC orientation. We first employed an isotopically symmetric bilayer (dDMPC) to distinguish the SFG signal of the 6-KC from that of the lipid bilayer. Figure 2 shows the ssp and psp spectra of the 6-KC after injecting different amounts of 6-KC into the subphase of the d-DMPC bilayer and reaching its equilibrium. The fitting curves (red lines) in Figures 2−5 are just used for guiding the eyes of readers and do not have any physical meaning. The spectra are dominated by several peaks at 2850, 2865, 2887, 2905, 2935, and 2967 cm−1. A strong peak at 2935 cm−1 in the psp spectrum is observed from the chiral methyl substitutions at the steroid ring. Possible assignments for those modes are given in Table S1 (Supporting Information) according to the Raman spectral features of cholesterol and SFG peak assignments.21−28,30 It is worth noticing that the ssp intensity from the methyl group of the 6-KC tail follows an almost linear dependence of the 6-KC concentration (Figure 2C), strongly suggesting that the number of the 6-KC increases linearly. However, the psp intensity from the chiral methyl substitutions reaches a plateau after a certain concentration (≥3 μL) (Figure 2D). Generally, the chiral signal may be contributed by the molecules in the bulk when SFG spectra are collected using transmission geometry.31 In this case, the chiral signal shows a quadratic dependence on the bulk concentration.31 However, it was also found that the bulk contribution can be negligible if the reflection geometry is employed.32 Thereby, the psp signal obtained here could be contributed from the interface, which is in fact confirmed by Figure 2D. In addition, the effect of the orientation on the chiral psp signal can be ignored because the orientation of the 6-KC molecular axis is independent of the 6-KC concentration, as shown below. Accordingly, the chiral signals depend mostly on the chiral arrangement of the chiral centers rather than the absolute number of them. According to the SFG symmetry constraints, the formation of tail to tail organization on both leaflets (Figure S1, Supporting Information) can effectively cancel the ssp signal. Similarly, the formation of α−α and β−β structures (a and b in Figure 1B) can effectively eliminate the chiral signal, but the occurrence of the α−β structures cannot.
Figure 1. (A) Molecular structure and the defined molecular axis of 6KC. (B) Three possible forms of side-by-side organizations of 6-KC.
show distinct dependence on the cholesterol concentration. We therefore realize that a combination of achiral-sensitive ssp and chiral-sensitive psp polarization measurements can permit explicit differentiation of the structure change of the lipid, cholesterol isooctyl tail, and methyl substitutions of cholesterol sterol rings, as well as membrane-bound water molecules. It will be able to provide detailed information about the assembling processes of cholesterol in the membrane, which has been difficult to acquire with other techniques. To materialize this idea, we used cholesterol analogue 6-ketocholestanol (6-KC) as
Figure 2. (A) The ssp intensity and (B) psp intensity change with the 6-KC amount. (C) The maximum ssp intensity at 2865 cm−1 and (D) psp intensity at 2935 cm−1 as a function of the 6-KC amount. 420
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collected from the d-DMPC bilayer/water interface at different times after adding 3 μL 6-KC solution. The ssp spectra are mainly contributed by the 6-KC achiral isooctyl tail and the water. Upon injecting the 6-KC solution into the subphase, the membrane is gradually dehydrated, resulting in an intensity decrease of water signals. To qualitatively determine the phase sign of CH2 and CH3 symmetric stretch modes, we have fitted the spectra in Figure 3A using eq S2 (Supporting Information). The fitting parameters are presented in Table S1 (Supporting Information). We acknowledge that such a fitting may not be accurate enough to describe the precise strength of CH2 and CH3 stretching modes because of the serious overlap in frequency. However, the relative phase of different modes can still be clearly determined. Generally, the symmetric stretch and Fermi resonance modes have the same phase sign, while the symmetric stretch and asymmetric stretch modes have opposite phase. It is found that the phase signs that give the best fitting are negative for the CH3 symmetric mode while positive for the CH2 symmetric mode and water O−H stretch mode. It means that the average orientations of 6-KC tailed methyl groups and the O−H dipole both point toward the CaF2 substrate, whereas the molecular plane of CH2 tilts away from the substrate plane. The O−H orientation direction is consistent with the phasesensitive SFG results.34 These results suggest that 6-KC molecules keep their methyl groups of the achiral tail toward the DMPC tail of the distal leaflet. It is actually energetically favorable because the primary driving force for the insertion of the cholesterol into the membrane is the hydrophobic interaction between its isooctyl tail and the lipid hydrocarbon chain. Earlier NMR study also supported that the methyl group at the hydrophobic tail of the cholesterol shows strong correlation with the terminal group of the DMPC chain portion.36 After obtaining the molecular pointing direction of the 6-KC, we then determined the tilt angle of the 6-KC molecular axis in lipid bilayers through the keto and C−OH groups. It has been shown that a single value measured from any of the rings is enough to determine the average orientation angle of the cholesterol molecular axis because of the rigid structure of the sterol ring.37 Here, the orientation of the 6-KC molecular axis θM can be deduced by the simple relationship θM = 180° − ∠CCO−θCO or θM ≈ θC−O. ∠CCO is the angle between the molecular axis and the axis of the CO bond and equals ∼120°. Using the methodology developed by Tyrode et al.,38 the orientation of CO bond can be determined by collecting SFG spectra with ssp and ppp polarization combinations. The (2) relation between the χ(2) ppp/χssp ratio and θCO is presented in Figure S2 (Supporting Information). Figure 3B shows the ssp and ppp spectra in the CO region. All of the ssp and ppp spectra give one strong peak at ∼1710 cm−1 and two shoulder peaks at ∼1680 and 1730 cm−1, respectively. The ∼1680 and 1710 cm−1 peaks originate from 6-KC molecules, as confirmed by the corresponding infrared spectra (Figure S3, Supporting Information) and SFG spectra of pure 6-KC (Figure S4, Supporting Information). The 1730 cm−1 signal is generated by the carbonyl groups of the lipid bilayer. A detail on the assignments of these peaks is given in the Supporting (2) (2) /χssp Information. Here, the experimentally measured χppp −1 ratio of the peak at 1710 cm is 0.92, yielding a tilt angle θCO of about 50° with respect to the surface normal when a δdistribution is assumed. The orientation angle of the molecular axis θM is then calculated to be ∼10°. This value agrees well with the results deduced from SFG spectra of the C−OH group
As a result, the dependence of the ssp and psp intensity on the 6-KC amount strongly indicates that the tailed methyl groups of different 6-KC molecules adopt the same orientation direction, while the chiral methyl substitutions organize with the α−β structure at low 6-KC concentration and mainly α−α or β−β structures at high 6-KC concentration. Even though SFG experiments do not permit to distinguish α−α and β−β structures, the formation of β−β structure apparently helps to enhance the favorable van der Waals interactions between the smooth α face of sterol rings and the acyl chains of the lipid and thus facilitates the maximal molecular packing. Therefore, the 6-KC more likely adopts the β−β structure at high 6-KC concentration. Such β−β organization is consistent with the conclusions made in previous NMR studies.33 Future studies by X-ray and molecular dynamics simulation may provide more detailed insight into the 6-KC structures at the interface. To get a precise molecular description of cholesterol’s behavior in the membrane, it is necessary to determine the orientation of the 6-KC in the lipid bilayer. The molecular orientation of a functional group can be obtained by relating SFG susceptibility χijk(i,j,k = x,y,z) to the molecular hyperpolarizability βlmn(l,m,n = a,b,c).20−22 If the relation between the χijk(i,j,k = x,y,z) value and the orientation angle (θ) is given, θ can be deduced by measuring the ssp and ppp spectral intensity ratio. In addition, the orientation can also be qualitatively determined in terms of the phase of the vibrational modes.20−22 Here, we first determined the dipole direction of the methyl group at the 6-KC tail. Previous phase-sensitive SFG study indicated that membrane-bound water molecules are oriented preferentially by the electrostatic potential imposed by the phospholipids.34 In our system, the interfacial water SFG signal is dominated by the water molecules that are present at the boundary between the outer bilayer leaflet and the bulk water, rather than the confined water between the lipid bilayer and CaF2 prism surface.35 Therefore, it is possible to determine the dipole pointing direction of the methyl group at the 6-KC tail from its relative phase sign with respect to the water O−H stretch orientation. The relative phase sign of the water O−H stretch near the DMPC lipid has been determined by a previous phase-sensitive SFG study.34 Here, we use this value directly in our fitting procedure. Figure 3A plots the ssp spectra
Figure 3. (A) The ssp spectra collected from the d-DMPC bilayer/ water interface at different times after adding 3 μL 6-KC solution. (B) The ssp and ppp spectra in the CO region after injecting 3 μL 6-KC solution into the subphase (∼2.0 mL) of the lipid bilayer and reaching its equilibrium. 421
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(Figure S7, Supporting Information). The ppp spectra for the C−O bond show a peak at 1050 cm−1, and no signal is observed in ssp spectra, suggesting that θC−O is quite small (θC−O < 15°). In addition, the orientation angle is independent of the 6-KC concentration. Our result agrees well with previous studies by X-ray diffraction,39 permeability, and binding measurements,40 which indicate that the 6-KC is oriented nearly perpendicular to the plane of the bilayer with both the keto and hydroxyl groups located near the hydrocarbon−water interface. Molecular dynamics simulations also show that the orientation of the free-energy minima for the cholesterol in the phospholipid bilayers has a tilt angle of 17−28°.41 Following the addition of the 6-KC, we monitored the psp SFG signal at 2935 cm−1 and ssp signal at 1710 cm−1. Figure 4 Figure 5. (A) Time-dependent ssp spectra of the d-DMPC bilayer after 3 μL 6-KC solution was injected into the subphase of the lipid bilayer. (B) Time-dependent ssp spectra intensity at 2075 cm−1. (C) Time-dependent fitting strength of the CD3 symmetric stretch.
analyzing the SMD change of the d-DMPC molecules in the monolayer at the air surface and the bilayer at the water interface. Here, the SMD is equal to zero for the bilayer before injecting 6-KC molecules because no SFG signal at 2075 cm−1 is observed. Because the orientation angle (θCD3) of the terminal methyl group in the d-DMPC can be deduced in terms (2) of the relationship between the ratio of χssp (CD3,as)/ (2) χssp (CD3,ss) and θCD3 (Figure S9, Supporting Information),42 a change in SMD can be isolated from the monolayer at the air surface and the bilayer at the water interface after injecting 6KC solution. Here, the Fresnel factor at the air surface is about 1.16 times compared to that at the water interface for our experimental geometry. For the case of injecting 3 μL 6-KC (Figure 5 and Figure S10, Supporting Information), the value of χ(2) ssp (CD3,ss) of the monolayer at the air surface is estimated to be about 1.07 times compared to that of the bilayer at the water interface according to the orientation angle change (Figure S9, Supporting Information). By comparing the spectral intensity, we may conclude that SMD of the terminal methyl CD3 group that contributed to the SFG signal for the 6KC bilayer system at the water interface is about 75% of the density for the monolayer at the air surface. This is based on the fact that the intensity of the 2075 cm−1 peak after the interaction reaching its equilibrium is about 85% of the intensity of the monolayer at the air surface. If the flip-flop of dDMPC molecules takes place in the presence of 6-KC, the number of the terminal methyl CD3 group that contributes to the SFG signal at 2075 cm−1 in bilayer environments will be much less than the number of the monolayer at the air surface. Furthermore, the intensity of the 2075 cm−1 peak should depend on the addition of the 6-KC amounts. These expected observations are completely opposite to what has been observed in our experiments. Instead, the intensity of the 2075 cm−1 peak is found to be independent of the addition of the 6-KC amounts (≥3 μL, Figure S10, Supporting Information) and remains stable more than 500 min. These results, taking into account the fact that the DMPC showed a very fast flip-flop, with a flip-flop half-life (t1/2) of 1.3 min at 20.4 °C,19 suggest that the flip-flop of the DMPC is inhibited in the presence of the 6-KC. In this case, the 6-KC molecules are located at the outer leaflet of the lipid bilayer and occupy the dDMPC position at the outer leaflet. Thus, the signals of CD3 generated from both leaflets cannot cancel each other.
Figure 4. (A) Time-dependent psp spectra intensity at 2935 cm−1 and (B) ssp intensity at 1710 cm−1 following addition of 3 μL 6-KC.
shows the time-dependent psp intensity of the 6-KC methyl group at 2935 cm−1 and the ssp intensity of the keto group at 1710 cm−1 after injecting 3 μL 6-KC solution into the subphase of the lipid bilayer at t = 0 min. The psp signal at 2935 cm−1 and ssp signal at 1710 cm−1 both increase quickly to a plateau in 20 min and become stable. To investigate the effect of the 6KC diffusion, we performed another set of experiments: we prepared the d-DMPC monolayer on CaF2 prisms first using the Langmuir−Blodgett method and then contacted the dDMPC/6-KC mixture at the water/air surface (with mol ratio of 1:2) to form a bilayer. The time-dependent psp signal at 2935 cm−1 (Figure S8, Supporting Information) shows that it takes less than 20 s for the interaction to reach its equilibrium when the diffusion of the 6-KC is not required, indicating a rapid interaction between the 6-KC and the lipid bilayer. The results are further confirmed by the time-dependent ssp spectra of the d-DMPC bilayer in the frequency range of 2000−2300 cm−1 (Figure 5) after injecting 3 μL 6-KC solution into the subphase of the lipid bilayer at t = 0 min. Before injecting 6-KC molecules, no SFG signal at 2075 cm−1 was observed. The peak at 2075 cm−1 originates from the symmetric stretch of the CD3 group.22 The absence of the CD3 signal from the bilayers suggests that the proximal leaflet and distal leaflet have very similar structures, and the signals generated from both leaflets can effectively cancel each other. However, the injection of the 6-KC can break the inversion symmetry on the lipid bilayer, resulting in the appearance of SFG signal from CD3 vibrational modes. The signal at 2075 cm−1 increases quickly and reaches a plateau in 20 min after injecting 6-KC molecules. All of these results indicate that the flip-flop motion of the 6-KC in the membrane takes time to occur, which is further confirmed by the analysis given below. It is well-known that the SFG signal intensity is closely related to the surface molecular density (SMD), the average orientations, and Fresnel coefficients. Therefore, the flipping of the d-DMPC and the 6-KC can roughly be estimated by 422
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The Journal of Physical Chemistry Letters The power and uniqueness of combining achiral- and chiralsensitive polarization measurements to probe the organization and transport of the cholesterol in membranes are nicely demonstrated in this study. We have unambiguously identified that 6-KC molecules are aligned at the outer leaflet of the lipid bilayer with a tilt angle of about 10°. The 6-KC adopts the α−β structure at low 6-KC concentration and most likely the β−β structure at high 6-KC concentration. This detailed structural information allows us to validate different structural models proposed in the literatures. It can be seen that the long-range ordering as assumed in the superlattice model9 is not always necessary. The condensed complex model, which accurately describes cholesterol’s behavior in the monolayer by the formation of stoichiometric complexes between cholesterol and lipids,3,8 fails to match our experimental observations. The umbrella model can explain well the organization of the 6-KC structure in the DMPC bilayer. The CD3 SFG signals from the d-DMPC imply that the presence of a frustum-shaped cluster of the 6-KC at the outer leaflet of the lipid bilayer occupies the dDMPC place. In this case, the hydroxyl head groups of the clustered 6-KC gather together to form the top of the frustum and to reduce the water penetration between 6-KC groups. The cluster formation of the 6-KC apparently hinders the occurrence of the flip-flop. To conclude, the method employed in this study has permitted clarification of the 6-KC organization, conceptual structural models for the cholesterol−lipid interactions, as well as the flip-flop rate of 6-KC in a lipid bilayer. It will be used as a unique and effective optical marker to characterize detailed structural information of cholesterol associated with the lipid bilayer in situ in different chemical environments. The cell membrane is a complex system containing many compositions. Other components may play important roles in cholesterol flipping, inducing the cholesterol distribution in both leaflets of the bilayer. Our method will be able to elucidate the dynamics of cholesterol flipping and assembling in a real cell environment in situ in the future.
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ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the National Basic Research Program of China (Grant 2010CB923300) and the National Natural Science Foundation of China (Grants 21273217, 91127042, 20925311, and 21161160557).
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ASSOCIATED CONTENT
S Supporting Information *
Details about materials, the experimental procedure, SFG data analysis, Figures S1−S10, including forms of 6-KC, the relation (2) between the χ(2) ppp/χssp ratio and different θCO, infrared spectra of 6-KC, ssp and ppp spectra in the CO region, the molecular structure of d-DMPC, ssp spectra of 6-KC in the CO region in the absence and presence of lipid bilayer, ssp and ppp spectra of C−OH stretching, time dependence of the psp spectra intensity, relations between the χ(2) ssp (CD3,as)/ (2) χ(2) ssp (CD3,ss) and θCD3 and the χssp (CD3,ss) and θCD3, and the ssp SFG signal of lipid monolayer and bilayer, and Table S1 showing the fitting parameters for the ssp spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions #
S.M. and H.L. contributed equally.
Notes
The authors declare no competing financial interest. 423
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Sum Frequency Generation Spectroscopy. Langmuir 2006, 22, 5341− 5349.
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dx.doi.org/10.1021/jz402537w | J. Phys. Chem. Lett. 2014, 5, 419−424
