Effect of Ca2+ to Sphingomyelin Investigated by Sum Frequency Generation Vibrational Spectroscopy

Article

Effect of Ca2D to Sphingomyelin Investigated by Sum Frequency Generation Vibrational Spectroscopy Rong-juan Feng,1,2 Lu Lin,1,3 Yi-yi Li,1,2 Ming-hua Liu,3,4 Yuan Guo,1,2,* and Zhen Zhang1,* 1 Beijing National Laboratory for Molecular Sciences, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China; 2University of Chinese Academy of Sciences, Beijing, China; 3National Center for Nanoscience and Technology, Beijing, China; and 4Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Colloid, Interface and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China

ABSTRACT The interactions between Ca2þ ions and sphingomyelin play crucial roles in a wide range of cellular activities. However, little is known about the molecular details of the interactions at interfaces. In this work, we investigated the interactions between Ca2þ ions and egg sphingomyelin (ESM) Langmuir monolayers at the air/water interface by subwavenumber high-resolution broadband sum frequency generation vibrational spectroscopy (HR-BB-SFG-VS). We show that Ca2þ ions can induce ordering of the acyl chains in the ESM monolayer. An analysis of the one alkyl-chain-deuterated ESM revealed that the Ca2þ ions do not affect the N-linked saturated fatty acid chain, although they make the sphingosine backbone become ordered. Further analysis of the SFG-VS spectra shows that the interactions between ESM and Ca2þ ions make the orientation of the methyl group at the end of sphingosine backbone change from pointing downward to pointing upward. Moreover, a large blue shift of the phosphate group at the CaCl2 solution interface indicates, to our knowledge, new cation binding modes. Such binding causes the phosphate moiety to dehydrate, resulting in the conformation change of the phosphate moiety. Based on these results, we propose the molecular mechanism that Ca2þ ions can bind to the phosphate group and subsequently destroy the intramolecular hydrogen bond between the 3-hydroxyl group and the phosphate oxygen, which results in an ordering change of the sphingosine backbone. These findings illustrate the potential application of HR-BB-SFG-VS to investigate lipidcation interactions and the calcium channel modulated by lipid domain formation through slight structural changes in the membrane lipid. It will also shed light on the interactions of complex molecules at surfaces and interfaces.

INTRODUCTION Sphingolipids are one of the most important components of the plasma membranes of cells. These molecules act as mediators of signaling pathways that regulate cell growth, differentiation, multiple cell functions, cell apoptosis, and cellular calcium homeostasis (1–3). Sphingomyelins (SMs, N-acyl-sphingosine-1-phosphorylcholine) are the most common sphingolipids in eukaryotic cellular membranes and neuronal tissues (4). It has been reported that SM molecules along with cholesterol play a critical role in the formation of microdomains in biomembranes and lipid rafts (5–7). In addition, SM lipids are thought to have important biological functions related to membrane signaling and protein trafficking (8). Because of the hydroxyl group of the long sphingosine chain base, phosphate oxygen atoms of

Submitted January 20, 2017, and accepted for publication April 17, 2017. *Correspondence: [email protected]; [email protected] Editor: Elsa Yan. http://dx.doi.org/10.1016/j.bpj.2017.04.026

the headgroup, and amide groups of the long acid chain base, SM can readily form extensive intra- and intermolecular hydrogen bonds in membranes (9–11), causing SMs to have a relatively strong tendency for domain formation (12–15). Natural SMs usually contain a sphingosine backbone and an acyl chain of unequal length. The backbone possesses the 4,5-trans double bond, and the N-linked acyl chain is fully saturated and has a length of up to 30 carbon atoms (4,16). The interactions between Ca2þ ions and membrane lipids, particularly SM lipids, are related to the induction of diverse cellular activities, including metabolism, cell growth, differentiation, membrane fusion, and cell death (17). Therefore, increasing attention has been paid to the interactions of SM with Ca2þ ions (16,18–22). In the 1960s, Shah and Schulman (18) used surface pressure and surface potential measurements to investigate the interactions of SM with Ca2þ ions. They found that the Ca2þ-SM interaction is influenced by the steric hindrance of the 3-hydroxyl group (3OH) that

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is vicinal to the phosphate group. Several studies of the interactions of Ca2þ ions with SM bilayers performed by NMR and Fourier transform (FT)-Raman spectroscopy have reported that Ca2þ ions mainly bind to the phosphate group in the headgroup and do not change the conformation of the O–C–C–Nþ backbone (19–21). Rujoi et al. (16) investigated the effect of Ca2þ ions on SM structures using FT infrared (FT-IR) spectroscopy and concluded that Ca2þ ions not only bind to and/or dehydrate the phosphate moieties but they also strengthen the intermolecular hydrogen bonds. Sugimori et al. (23) used density functional theory calculations to investigate the interactions, and they showed that Ca2þ ions prefer to interact with the choline moiety near the phosphate group. However, previous studies have mainly focused on the interactions of Ca2þ ions with the headgroup, and the conformational and orientational changes of the two hydrophobic chains have not been investigated. It is critical to investigate the possible changes in the conformation and orientation of the saturated fatty acid and sphingosine chains because these changes are related to the lipid-ion channel and the functions of membranes (24–30). The sum frequency generation (SFG) technique has been shown to be a powerful tool because it is sensitive to the structure, structural variations, and the complex local environment at the interface, and it has been used to determine the functional group orientation and conformation structure of lipid monolayers at interfaces (26,31–33). To better understand the molecular structure at interfaces, different polarization conditions have also been used to identify the orientation of molecules at interfaces (34,35). The principle of the SFG experiments is described briefly in the Supporting Materials and Methods. Although there have been many developments in the instrumentation and interpretation of SFG-vibrational spectroscopy (SFG-VS) data, the limitations of its application to different complex molecular sys-

tems, such as bimolecular surfaces and interfaces, still restrict the ability to interpret SFG-VS data (36–38). To overcome these limitations, high-resolution broadband SFG-VS (HR-BB-SFG-VS) with subwavenumber resolution (0.4 cm1) and a large signal-to-noise ratio (SNR) has been developed (39). In addition to the surface sensitivity, HR-BB-SFG-VS can obtain unambiguous interfering fine spectral features and nearly intrinsic spectral lineshapes of the molecular vibrational modes because of the high resolution and large SNR (40,41). To elucidate the effect of Ca2þ ions on the structures of the two hydrophobic chains, we used a combination of surface pressure measurements and HR-BB-SFG-VS to investigate the structures of egg SM (ESM, as shown in Fig. 1) lipid monolayers at pure water and 2 M CaCl2 aqueous solution interfaces. Such studies have rarely been carried out, except for the study of SMs interact with cholesterol at the air/solid interface by Weeraman et al. (33). The concentration of 2 M CaCl2 in the solution is relevant in the biological study and biological systems. For example, 2 M CaCl2 solutions can be used during the cell competence in the laboratory (42). In addition, the study of high concentration salt effect is a common method to understand the essence of the ion-lipids interactions (26). To observe the subtle effect of Ca2þ ions on the organization of the ESM monolayers, a relatively high Ca2þ concentration (2 M) was used in this study rather than <0.4 M, as used in previous studies (26,31,43). In this article, we reported that Ca2þ ions can bind to the phosphate group and subsequently destroy the intramolecular hydrogen bonds between the 3OH and the phosphate oxygen, which results in the ordering change of the sphingosine backbone and the change of orientation of the methyl group at the end of sphingosine backbone from downward to upward.

FIGURE 1 Given are molecular structures of (a) ESM and (b) ESM-d31. Both ESM and ESM-d31 consist of three parts: two hydrophobic acyl chains, one hydrophilic polar headgroup, and an interface region. To see this figure in color, go online.

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MATERIALS AND METHODS Materials Chicken ESM and chain-deuterated ESM (ESM-d31) were purchased from Avanti Polar Lipids (Alabaster, AL) and used as received. Chloroform and methanol (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO). ESM was dissolved in a methanol-chloroform (5:1, v/v) mixture to make a 0.5 mM solution. The stock solutions of SM were stored in the dark at 20 C and warmed to ambient temperature before being used. The molecular structures of ESM and ESM-d31 are shown in Fig. 1. Before its use, calcium chloride dehydrate (ACS reagent grade, R99%; Alfa Aesar; Haverhill, MA) was baked at 550 C for 5 h under argon protection conditions to remove potential organic contaminants. The baked calcium chloride was then used to prepare a stock solution by dissolving in pure water (18.2 MU,cm, pH 5.6). No C–H stretching features (2800–3000 cm1) of organic contaminant were observed in the SFG-VS spectrum of the calcium chloride stock solution following a previously published procedure (44).

Surface pressure-area isotherm The surface pressure-area (p–A) compression isotherms were measured by a computer-controlled Langmuir trough (Atotal ¼ 273 cm2; KSV Mini Trough; Biolin Scientific, Espoo, Finland). The trough was made of Teflon and equipped with two compression barriers made of Delrin (DuPont, Wilmington, DE). The surface pressure of the Langmuir monolayer was monitored by a Wilhelmy plate made of platinum and flamed with a Bunsen burner before use. The trough and barriers were rinsed with ethanol and Millipore water (Millipore, Billerica, MA) several times before the measurements. The whole trough was enclosed in a homebuilt Plexiglas protective box to eliminate dust and other possible airborne contaminants. Before spreading the SM solution on the pure water or CaCl2 aqueous solution, the aqueous surface was swept by the barriers to ensure surface cleanliness, and the cleanliness was confirmed when the final surface pressure was <0.2 mN/m. When preparing the Langmuir monolayers, an appropriate volume of the SM solution was spread on the water or CaCl2 aqueous surface with a microsyringe in a dropwise manner. After waiting for 15 min to allow the solvent to evaporate, the films were compressed with a constant rate of 10 mm/min. During this process, the surface pressures and the mean molecular areas were continuously monitored by compressing the barriers.

Sub-1 cm1 resolution broadband SFG-VS A recently developed HR-BB-SFG-VS with subwavenumber spectral resolution can provide an accurate spectral lineshape and allow direct probing of the molecular interactions by SFG-VS spectra (37,39). The HR-BBSFG-VS spectrometer used in this study was set up by our group. It consists of two sets of synchronized Ti:sapphire oscillators/amplifiers. One is a Ti:sapphire regenerative amplifier (Spitfire Ace; Spectra-Physics, Mountain View, CA) seeded with an oscillator (Mai Tai; Spectra-Physics) and pumped with two diode-pumped green lasers (Empower; Spectra-Physics). The amplifier is used to generate laser pulses centered at 800 nm with a fullwidth at half-maximum (FWHM) of 30 nm and a pulse duration of 35 fs. The amplifier produces 7.5 mJ energy per pulse with a repetition rate of 1 kHz. Two commercial optical parametric amplifiers (TOPAS-Prime; Spectra-Physics) are pumped with 2.5 and 5.0 mJ of the amplified 800 nm beam, respectively. For one TOPAS-Prime amplifier, the signal and idler are used in a difference frequency mixing process on a silver gallium disulfide (AgGaS2) crystal, resulting in 10 mJ IR pulses that are tunable around a central wavelength of 3440 nm (2900 cm1) with a FWHM of 400 cm1, and the other 5 mJ TOPAS-Prime amplifier results in a 8 mJ pulse that is tunable around a central wavelength of 9540 nm (1050 cm1) with a FWHM of 300 cm1, which are used as IR pulses.

The other synchronized picosecond laser system is described below. The master pulse train originates from a 35 fs oscillator (Mai Tai; Spectra-Physics) and its repetition rate (84 MHz) is used as the frequency reference for stabilization of a 50 ps oscillator (Tsunami; Spectra-Physics). The 50 ps oscillator is pumped with a diode-pumped green laser (Empower; Spectra-Physics). The amplifier (Spitfire Ace; Spectra-Physics) is used to generate laser pulses centered at 800 nm with a pulse duration of 50 ps, which are used as visible pulses. It produces 2.0 mJ energy per pulse with a repetition rate of 1 kHz. The stability and pulse width of the 50 ps pulse are monitored with a homebuilt autocorrelator throughout the whole experiment. The manually synchrolocked controls provide a few nanoseconds of continuous relative time delay between the slave and the master pulses. The infrared and visible laser beam temporally and spatially overlap at the air/liquid interfaces, aligning in a copropagating geometry with an IR incident angle of 55 and a visible incident angle of 63 relative to the surface normal. The typical acquisition time of a single SFG-VS spectrum in a specific polarization combination is 30 min using a monochromator (model No. SP2750; 750 mm, 1800 lines/mm grating; Princeton Instruments, Trenton, NJ) and electron-multiplied charge-coupled device camera cooled at 70 C (ProEM EM-charge-coupled device camera; 1600  400; Princeton Instruments). With all the instruments, the spectral resolution of the sub-1 cm1 HRBBSFG-VS spectrometer was characterized as 0.4 cm1 in our setup. The SFG-VS spectra were recorded in the C–H stretching region (2800– 3000 cm1) and P–O stretching region (1000–1200 cm1) in the ssp and ppp polarization combinations (SFG, visible, and the IR laser beams, respectively). Each SFG-VS spectrum was processed by subtracting the background and blocking the IR pulses, and then normalized to the SFG-VS signal of thick z-cut quartz (12.5  12.5  5 mm) in the 2800–3000 cm1 range and a GaAs(110) wafer in the 1000–1200 cm1 range to remove spectral distortion caused by the energy profile of the IR pulse. To minimize the laser heating effects on the spectrum of the ESM monolayer at the interface, a Teflon (Chemours, Wilmington, DE) dish was placed on a rotation stage (model No. PR50CC; Newport, Stratford, CT) operated by a motion controller (model No. XPS-Q4; Newport) with a fixed rate of 1200 /s. All of the measurements were performed at controlled room temperature (22.0 5 0.5 C) and humidity (20%).

RESULTS AND DISCUSSION Surface pressure-area isotherm of ESM The p–A isotherms of ESM on pure water and CaCl2 solution subphases are shown in Fig. 2. With decreasing mean molecule area (MMA), the isotherm of ESM on pure water exhibits the following phases: gas phase (G), liquidexpanded (LE), LE phase to the liquid-condensed phase (LE–LC coexistence phase), and LC phase. This phase behavior is consistent with that previously reported (33,45–47). Compared with the pure water interface, the ESM isotherm shifts to higher MMA at all surface pressures at the CaCl2 solution interface. This shift can be attributed to binding of Ca2þ ions to the negatively charged moiety (PO2) of ESM, which leads to increased repulsive interactions between ESM molecules and a larger surface area for each ESM molecule. Similar observations have been reported in previous studies (31,43). It is worthwhile to point out that the addition of 2 M CaCl2 to the aqueous subphase causes the domain formation in the SM monolayer. This is consistent with previous reports (17,48). The SFG spectra of the SM monolayer are an average of two different coexisting phases.

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˚ 2) isoFIGURE 2 Given here are the surface pressure (mN/m)-area (A  therms of ESM Langmuir monolayers at 24 C at the pure water (red solid line) and CaCl2 solution (blue dashed line) interfaces: G, gas phase; LE, liquid-expanded phase; LC, liquid-condensed phase; and LE-LC, the coexistence of LE and LC. After adding Ca2þ ions into the subphase, the ESM isotherm shifts to higher MMA, indicating that Ca2þ ions bind to the phosphate group of ESM. The vertical arrows and dashed lines denote the surface area in the HR-BB-SFG-VS measurements in subsequent experiments. The isotherm curves shown here are the averages of three measurements. To see this figure in color, go online.

Ordering effect of Ca2D ions on the acyl chains To obtain the effect of Ca2þ ions on the conformation of the hydrocarbon chains, HR-BB-SFG-VS spectra were recorded in the C–H stretch region. The spectra were taken at two ˚ 2 per molecule in the LE– different molecule areas: 68 A 2 ˚ per molecule in the LC phase. Fig. 3 LC phase and 52 A shows the HR-BB-SFG-VS spectra of ESM monolayers at the pure water and 2 M CaCl2 solution interfaces under ssp and ppp polarization combinations, in which nine vibrational modes can be observed. All the spectra are fitted using Lorentzian lineshape functions; the detailed description can be found in the Supporting Materials and Methods. The HRBB-SFG-VS spectra identify a number of additional peaks, for example, more CH2 ss vibration modes, which are obscured in the lower resolution SFG-VS spectra (41,49,50). The comparisons of HR-BB-SFG-VS (spectral resolution 0.4 cm1) and the EKSPLA picosecond scanning system (spectral resolution 6 cm1) are shown in Fig. S3. The peaks centered at 2874, 2926, 2942, 2953, and 2963 cm1 can be assigned to CH3 symmetric stretching (CH3 ss), CH2 Fermi resonance, CH3 Fermi resonance, CH2 ss in the headgroup, and CH3 asymmetric stretching (CH3 as), respectively (26–28,30,32,33,51–56). The exact assignments of all of the peaks are beyond the scope of this article. The peaks centered at 2840–2860 cm1 should belong to the symmetric stretching mode of CH2 groups. The fitting results are listed in Table S1, and the fitted peak positions are labeled with vertical dashed lines in

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FIGURE 3 Shown here are HR-BB-SFG-VS spectra of ESM monolayer ˚ 2/molecule films on the pure water and CaCl2 solution subphases at (a) 68 A ˚ 2/molecule under ssp and ppp polarization. The solid lines are and (b) 52 A the global fitting curves using the Lorentzian lineshape, and the vertical dashed lines denote the peak positions from the fitting. To see this figure in color, go online.

Fig. 3 a. As recently reported, to our knowledge the new CH2 spectral features observed here overlap with CH3-ss, CH3-as, or Fermi resonance in common BB-SFG-VS, but they can be well resolved by, to our knowledge, the new HR-BB-SFG-VS technique (49). It is worth noting that, in principle, each ESM molecule has an even number of CH2 groups and they should pairwise cancel the SFG-VS signal in the straight chain, but this is not the case in the real SFGVS measurements here. This may be because multiple microscopic structures with gauche defects can contribute to the CH2 SFG-VS spectrum. The CH2 groups connected to the terminal CH3 group and the CH2 groups linked to the serine group of the headgroup also contribute to the CH2 SFG-VS spectrum. In Fig. 3, the ratio of the intensities of the CH3 ss (2874 cm1) and CH2 ss (2850 cm1) modes for ˚ 2/molecule is larger than that for 68 A ˚ 2/molecule, indi52 A cating that the conformation of the ESM acyl chains for

Effect of Ca2þ to Sphingomyelin

˚ 2/molecule is more ordered, which is in agreement with 52 A previous studies of lipids (26–28,53,54,57–59). Compared with the spectra at the pure water interface, the CH3 ss peaks in the spectra at the 2 M CaCl2 solution interface are stronger, whereas the CH2 ss peaks become slightly weaker. These results indicate that the acyl chains of ESM are also more ordered at the 2 M CaCl2 solution interface under the same MMA. As shown in Fig. 1, ESM has two hydrophobic acyl chains—one is sphingosine backbone, and the other is N-linked fully saturated acyl chain. It is crucial to understand which acyl chain plays the main role in determining the lipid-ion interactions. Although previous studies have suggested that the N-linked acyl chain is important for determining the interaction of SM with cholesterol because intermolecular hydrogen bonds can be formed between cholesterol and the N-linked acyl chain (60–62), there is no direct research on the interaction between the two different acyl chains of ESM and Ca2þ ions. To investigate how the two hydrophobic acyl chains interact with Ca2þ ions, SFG-VS spectra of ESM-d31 (the structure shown in Fig. 1 b) were collected in both the C–H and C–D regions. The detailed effects of the two hydrophobic acyl chains will be discussed below. Effect of Ca2D ions on the two acyl chains Fig. 4 shows the ssp spectra of ESM-d31 monolayers with different MMA on water and the CaCl2 solution in the C–D stretching region. The ssp peaks at 2068, 2104, 2120, and 2219 cm1 have been previously assigned to the CD3 symmetric stretch (CD3 ss), the CD2 asymmetric stretch (CD2 as), CD2 Fermi resonance, and the CD3 asymmetric stretch (CD3 as), respectively (29,57,58). The peak at 2068 cm1 can also be assigned to CD3 ss, similar to what was observed in the C–H stretching region. In Fig. 4, there is an extra peak at 2056 cm1 in the ssp spectra. We do not assign this peak here because of the low SNR, but this will not change any of the conclusions of this study. It is worth noting that the CD2 Fermi resonance spectral shift in Fig. 4 has not been observed; instead, a slight intensity change of the CD2 Fermi resonance has been observed, which may result from the slightly different molecular vibrational coupling of the CD2 groups of the N-linked acyl chain in the presence of Ca2þ and in the absence of Ca2þ. As shown in Fig. 4, a and b, apart from the CD2 Fermi resonance, the ssp spectra of ESM-d31 on the water and CaCl2 solution subphases are almost identical in the C–D stretching region, indicating that Ca2þ ions have a negligible effect on the N-linked fully saturated acyl chain of ESM. This indicates the CH2 groups in the N-linked fatty acid acyl chain are well ordered and adopt the all-trans conformation on the water subphase, and the addition of Ca2þ ions does not affect the highly ordered N-linked alkyl chain.

FIGURE 4 Shown here are the HR-BB-SFG-VS ssp spectra of ESM-d31 monolayer films on the pure water and 2 M CaCl2 solution subphases at (a) ˚ 2/molecule (solid lines are fitting results using Lorentzian lineshape the 68 A ˚ 2/ functions, the fitting parameters are listed in Table S2) and (b) the 52 A molecule in the C–D stretching region. Compared with the spectrum on the water subphase, the spectrum on the 2 M CaCl2 solution subphase is almost the same, except for the CD2 Fermi resonance. The solid lines are the global fitting curves using the Lorentzian lineshape, and the vertical dashed lines denote the peak positions from the fitting. To see this figure in color, go online.

In contrast to the C–D stretching region, the SFG-VS spectra of the ESM-d31 monolayers on the pure water and CaCl2 solution subphases show a significant difference in the C–H stretching region, as shown in Fig. 5. For the same MMA, the CH3 ss intensity of the ESM-d31 monolayer on the CaCl2 solution subphase is larger than that on the water subphase, similar to that of ESM monolayers upon addition of Ca2þ ions into the subphase. Combined with the SFG-VS spectra of ESM-d31 in the C–D stretching region, it can be inferred that Ca2þ ions only influence the conformation of the sphingosine backbone. The different effects of Ca2þ ions on the two chains originates from the intramolecular hydrogen bonding between 3OH and the phosphate oxygen in the sphingosine backbone (23,63–66). The intramolecular hydrogen bonding, along with the rigid C4–C5 trans double bond, makes the sphingosine backbone tilt at a large angle with respect to the surface normal (61).

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Reverse orientation of the terminal methyl group with Ca2D ion addition As discussed above, in the spectra of ESM monolayers, the CH3 ss peak contains contributions from both the N-linked acyl chain and the sphingosine backbone. In contrast, in the spectra of the ESM-d31 monolayers, the CH3 ss peak solely originates from the sphingosine backbone. By comparing Figs. 4 and 5, we can easily conclude that the N-linked acyl chain remains unaltered and the sphingosine backbone becomes more ordered upon addition of Ca2þ ions. With this information, if we assume that the changes in the intensities (DI) of the CH3 ss peaks from the sphingosine backbone are the same for both the ESM and ESM-d31 monolayers by adding Ca2þ, then  > R ; R (1) I

ESMd31 ;Ca2þ IESMd ;H O 31 2

FIGURE 5 Shown here are the HR-BB-VSFG ssp and ppp spectra of ESM-d31 monolayers on the pure water and 2 M CaCl2 solution subphases ˚ 2/molecule in the C–H stretching region. The overwith (a) 68 and (b) 52 A all spectral amplitudes on the CaCl2 solution subphase are larger than that on the water subphase. The solid lines are the global fitting curves using the Lorentzian lineshape. To see this figure in color, go online.

Therefore, ESM molecules occupy a large molecule area at the air/water interface. At the CaCl2 solution interface, Ca2þ ions can easily bind to the phosphate moiety and imbed between 3OH and the phosphate oxygen because of their high binding affinities (23,67–69). As a result, the intramolecular hydrogen bonds are destroyed and the sphingosine backbones tilt parallel to the surface normal, leading to a smaller molecular area. This is in agreement with previous molecular dynamics simulations that showed that Ca2þ ions remarkably reduce the palmitoyloleoylphosphatidylglycerol ˚ 2 (67). In addition, the molecule area from 68.2 to 50.6 A sphingosine backbones become more ordered after tilting parallel to the surface normal, so the CH3 ss peak intensity becomes stronger in the presence of Ca2þ ions. In the case of ˚ 2/molecule, there are the ESM monolayer with the 68 A more intramolecular hydrogen bonds compared with the ˚ 2/molecule. As a consequence, upon adding Ca2þ 52 A ions, the increase of the CH3 ss peak intensity is more pro˚ 2/molecule. To nounced in the ESM monolayer with the 68 A the best of our knowledge, this is the first study investigating the influence of Ca2þ ions on the acyl chains of ESM with HR-BB-SFG-VS measurements.

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I ESM;Ca2þ IESM;H O 2

where R represents the ratio of the CH3 peak intensity at the CaCl2 solution interface to that at the pure water interface. However, comparison of Fig. 5 with Fig. 3 shows that the ratio is actually smaller for the ESM-d31 monolayer for both molecule areas. This inconsistency can be interpreted by assuming that the terminal methyl groups in the sphingosine backbone have opposite orientations on the water and CaCl2 solution subphases, the detailed analysis are discussed below. It is known that the CH3 ss peak intensity is proportional to the square of the effective susceptibility of the methyl groups, c(2). For ESM monolayers on the water and CaCl2 solution subphases, 2 ð2Þ ð2Þ IESM;H2 Of cN þ cS; H2 O ; 2 (2) ð2Þ ð2Þ IESM;CaCl2 f cN þ cS; CaCl2 ; ð2Þ

ð2Þ

where cN and cS are the effective susceptibilities of the terminal methyl groups in the N-linked acyl chains and the sphingosine backbones, respectively. Similarly, for ESMd31 monolayers on the water and CaCl2 solution subphases, ð2Þ 2 IESM-d31 ;H2 Of cS; H2 O ; 2 (3) ð2Þ IESM-d31 ;CaCl2 f cS; CaCl2 : ð2Þ

ð2Þ

ð2Þ

ð2Þ

Denoting a ¼ cS;H2 O =cN and b ¼ cS;CaCl2 =cN , the ratio of the CH3 ss intensities of the ESM and ESM-d31 monolayers on the different subphases can be expressed as 1 þ a 2 IESM;H2 O ; ¼ 1 þ b IESM;CaCl2 (4) a 2 IESM-d31 ;H2 O ¼ : b IESM-d31 ;CaCl2 According to the fitting results listed in Table S1 and Table ˚ 2/molecule, S3, for the monolayers at the 68 A

Effect of Ca2þ to Sphingomyelin

2 1 þ a 2 ¼ 3:5 ; 1 þ b 1:1 2 a 2 2 ¼ : b 1:2

(5)

As mentioned above, the N-linked acyl chains are well ordered regardless of the subphase, so it is reasonable to assume that jaj < 1 and jbj < 1. By solving Eq. 4, we obtained a ¼ 0.45 and b ¼ 0.75. To explain the meaning of the above ð2Þ values, we modeled the cssp versus q-curves for the methyl group (see Fig. S2) using previously reported equations (34,70,71). Based on the observations in Fig. S2, the negative value of a indicates that the two terminal methyl groups in the N-linked acyl chain and the sphingosine backbone are oppositely oriented on the water subphase, whereas the positive value of b indicates that the orientations of the two terminal methyl groups on CaCl2 solution subphase are the same. Because the N-linked acyl chains are well-ordered and the methyl groups in the N-linked acyl chains always point upward, we suggest that the terminal methyl groups in the sphingosine backbones point downward on the water subphase and turn upward after adding Ca2þ ions into the subphase. The downward orientation of the terminal methyl groups in the sphingosine backbones can be attributed to the intramolecular hydrogen bonds and the gauche defects of the CH2 group connected to the terminal CH3 group (72). After adding Ca2þ ions into the subphase, destruction of the intramolecular hydrogen bonds and the rigidity of the double bond in the sphingosine backbone make the terminal methyl groups change their orientation to pointing upward. In principle, phase-sensitive measurements or the maximum entropy method can be used to obtain directly

the phase information. In our experiment, however, it is difficult to finish. Because the SFG signal of CH3 originates from the sum of the contribution of the CH3 group located at the same alky chains for ESM or ESM-d31, it is impossible to distinguish the partially reversed CH3 group with phasesensitive measurements or the maximum entropy method. Binding of Ca2D ions to P–O As discussed in Ordering Effect of Ca2þ Ions on the Acyl Chains, the PO2 group and 3OH in the sphingosine backbone can form an intramolecular hydrogen bond. After adding Ca2þ, the intramolecular hydrogen bond is completely disrupted because the strong proton acceptor of the phosphate group of ESM will chelate with Ca2þ. More evidence can be found in the P–O vibrational modes in the SFG-VS spectra. Fig. 6 shows the P–O stretching region of SFG-VS spectra of ESM monolayers at the pure water and 2 M ˚ 2/molecule under the ssp CaCl2 solution interfaces at 52 A polarization combination. Three peaks at 1051, 1080, and 1091 cm1 are observed in the spectrum of the ESM monolayer at the pure water interface. There is no doubt that the peak at 1051 cm1 can be assigned to the P–O ester stretch (73,74). The peak at 1080 cm1 has not been previously identified in conventional SFG-VS spectra with lower resolution (31,73). Moreover, it is well known that the red shift of the symmetric stretch of PO2 (PO2 ss) can be observed owing to the loss of electron density of the P–O bonds during the hydration process. The peak at 1080 cm1 exhibits a larger red shift relative to PO2 ss than that of the peak at 1091 cm1, so it can be assigned to the symmetric stretch of PO2.H2O. The peak at 1091 cm1 can be assigned

FIGURE 6 Shown here are the HR-BB-SFG-VS spectra of the ESM monolayers in pure water (red circle, higher intensity spectrum) and 2 M CaCl2 solution (blue triangle, lower intensity spectrum) subphases in the P–O stretching region. The solid lines are the global fitting curves using the Lorentzian lineshape. To see this figure in color, go online.

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to the symmetric stretch of PO2.3OH, which is an intramolecular hydrogen bond between PO2 in the headgroup and 3OH in the sphingosine backbone. This hydration change-induced frequency redshift has also been investigated by theoretical calculations (75). The SFG-VS spectrum of the ESM monolayer in the presence of Ca2þ is remarkably different from that at the pure water interface (Fig. 6). The ssp spectrum of the ESM monolayer on the 2 M CaCl2 solution also contains three distinctive peaks, in which the peak positions have greatly blue shifted to 1104, 1120, and 1131 cm1. The most interesting observation here is that there are two new peaks at 1120 and 1131 cm1 in the spectrum of ESM at the 2 M CaCl2 solution interface. Such large blue shifts (by 40 cm1) have not been observed in previous investigations of the interaction between Ca2þ ions (0.4 M) and dipalmitoylphosphatidylcholine monolayers at the air/water interface by SFG-VS measurements (31), possibly because of the higher bulk Ca2þ ion concentration (2.0 M) and better SFG-VS spectral resolution (0.4 cm1) in this study. In fact, the smaller blue shifts (8–12 cm1) have been observed, for example, by Casillas-Ituarte et al. (31), who have also observed a blue shift of the PO2 symmetric stretch as the Ca2þ interacts with dipalmitoylphosphatidylcholine at the SiO2 interface, where they found that the peak shifts from 1112 to 1120 cm1 for phospholipid-Ca2þ complexes at the air/SiO2 interface because of the slightly different hydration state owing to the interaction of the silica surface with the headgroup. Moreover, FT-IR and NMR studies have suggested that Ca2þ ions not only dehydrate the phosphate moieties but also bind to phosphate (16,22), and the subsequent dehydration of the phosphate group results in an increase in the frequency (blue shift of 10 cm1) of the PO2 symmetric stretch (31,58,76–78). In addition, to elucidate the Ca2þ-phosphate interaction, more ssp SFG spectra were obtained from the phosphate group of ESM monolayers at a different concentration of the CaCl2 solution interface. We found the phosphate peak continuously

shifts to the blue; more details can be found in Fig. S4. Here we will not focus on the concentration-dependent analysis. Further studies are underway (R.-j.F., Y.-y.L, L.-L. Lu, G.-C. Han, Y.-P. Yi, Y.G., and Z.Z., unpublished data). Considering all of the above information, the peak observed at 1104 cm1 can be assigned to the partially dehydrated PO2 ss (16,30,73,79–81). The peaks at 1120 and 1131 cm1 in the spectrum of ESM at the 2 M CaCl2 interface (Fig. 6) only present at the CaCl2 solution interface, thus those peaks can only be assigned to the Ca2þ-ESM complex. One possible interpretation of the two peaks is based on symmetry splitting (82,83). That is, when the conformational freedom of the phosphate group is reduced by Ca2þ ions binding to the negatively charged oxygen atoms of the phosphate moiety, the effective symmetry of the phosphate group is also lowered because of the intermolecular interactions (83). This reduction of symmetry may result in splitting of the degenerate modes by introduction of new bands (84). Moreover, the SFG-VS intensity of the PO2 symmetric stretch at the CaCl2 solution interface is lower than that at the pure water interface. Such an intensity decrease may originate from the more tilted orientation of the P–N dipole in the headgroup with respect to the monolayer, which has been described in theoretical and experimental studies (31,69,79,85–87). With all of the detailed information described above, the HR-BB-SFG-VS spectra of ESM at the pure water and CaCl2 solution interfaces in the P–O stretching region confirm that Ca2þ ions bond to the phosphate moiety. Such binding induces a large blue shift (40 cm1) of PO2, and two new features appear at 1120 and 1131 cm1 from the ESM-Ca2þ complex. Molecular mechanism of Ca2D interacting with ESM Based on the above discussion, a full molecular mechanism for Ca2þ interacting with ESM is proposed (Fig. 7). The

FIGURE 7 Given here is the molecular mechanism of Ca2þ ions interacting with the headgroup and hydrocarbon chains of ESM. After adding Ca2þ ions to the subphase, the headgroup first dehydrates and then reorients, the sphingosine backbones become more ordered, and the terminal methyl groups in the backbones change their orientation from pointing downward to pointing upward. To see this figure in color, go online.

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binding of Ca2þ to the phosphate moiety first dehydrates the headgroup and then induces reorientation of the polar headgroups. In this case, the intramolecular hydrogen bonds between the phosphate moiety and 3OH in the sphingosine backbones are destroyed, and the sphingosine backbones become more ordered. These processes finally make the terminal methyl groups in the backbones change their orientation from pointing downward to pointing upward. CONCLUSIONS HR-BB-SFG-VS is used to investigate ESM monolayers at air/aqueous interfaces. The results reveal a molecular mechanism of Ca2þ interacting with ESM. Ca2þ ions first bind to the phosphate group and then induce dehydration of the phosphate group and reorientation of the polar headgroups. Subsequently, intramolecular hydrogen bonds between the phosphate moiety and 3OH in the sphingosine backbones are destroyed in the presence of Ca2þ ions. The sphingosine backbones become more ordered and the terminal methyl groups in the backbones change their orientation from pointing downward to pointing upward. In contrast, the conformation of the N-linked acyl chain remains unaltered upon addition of Ca2þ ions in the subphase. This study provides basic information for studying SM membranes at the molecular level. Furthermore, it also shows the applicability of HR-BB-SFG-VS to study complicated lipid systems. Further studies are underway to investigate the effect of Ca2þ on ESM by molecular dynamics simulations.

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Supporting Materials and Methods, four figures, and three tables are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(17) 30442-3.

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15. Yeagle, P. L., and J. E. Young. 1986. Factors contributing to the distribution of cholesterol among phospholipid vesicles. J. Biol. Chem. 261:8175–8181.

Z.Z. and Y.G. designed the research project. R.-j.F. and Y.-y.L. performed the experiments. Z.Z., Y.G., R.-j.F., and L.L. analyzed the data. All authors discussed the results and commented on the manuscript.

ACKNOWLEDGMENTS We thank Dr. Yuki Nagata and Prof. Yuanping Yi for thoughtful discussions. Y.G. is grateful for funding from the Natural Science Foundation of China (NSFC) (grant No. 21227802) and Ministry of Science and Technology of China (MOST) (grant No. 2013CB834504). Z.Z. is grateful for funding from the Natural Science Foundation of China (NSFC) (grant No. 21503235) and the Institute of Chemistry, Chinese Academy of Sciences (ICCAS), for start-up funding to support this work.

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