Article pubs.acs.org/JPCC
Water Orientation at Ceramide/Water Interfaces Studied by Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy and Molecular Dynamics Simulation Aniruddha Adhikari,† Suyong Re,‡ Wataru Nishima,‡ Mohammed Ahmed,† Satoshi Nihonyanagi,†,§ Jeffery B. Klauda,∥ Yuji Sugita,*,‡ and Tahei Tahara*,†,§ †
Molecular Spectroscopy Laboratory, ‡Theoretical Molecular Science Laboratory and iTHES, and §Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ∥ Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, Maryland 20742, United States ABSTRACT: Lipid/water interaction is essential for many biological processes. The water structure at the nonionic lipid interface remains little known, and there is no scope of a priori prediction of water orientation at nonionic interfaces, either. Here, we report our study combining advanced nonlinear spectroscopy and molecular dynamics simulation on the water orientation at the ceramide/water interface. We measured χ(2) spectrum in the OH stretch region of ceramide/isotopically diluted water interface using heterodyne-detected vibrational sum-frequency generation spectroscopy and found that the interfacial water prefers an overall hydrogen-up orientation. Molecular dynamics simulation indicates that this preferred hydrogen-up orientation of water is determined by a delicate balance between hydrogen-up and hydrogen-down orientation induced by lipid−water and intralipid hydrogen bonds. This mechanism also suggests that water orientation at neutral lipid interfaces depends highly on the chemical structure of the lipid headgroup, in contrast to the charged lipid interfaces where the net water orientation is determined solely by the charge of the lipid headgroup.
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INTRODUCTION Lipid/water interfaces are important for various bio-relevant processes. The plasma membrane of a cell comprising lipid bilayers, cholesterols, and membrane proteins acts as a venue for innumerable biochemical events that have a direct bearing on human health.1 Despite its uniqueness and importance, the experimental means of investigating such interfaces, which are just a few nanometers in thickness, remain limited. One of the chief hindrances to such a study is the possible interference by the signals that originate from adjacent bulk phases. Vibrational sum frequency generation (VSFG) is a powerful nonlinear spectroscopic technique, in which the bulk signal does not appear under the dipole approximation, and is hence suited to study interfaces with molecular-level interface specificity.2,3 Furthermore, heterodyne-detected vibrational sum frequency generation (HD-VSFG) offers several major advantages over the conventional (homodyne-detected) VSFG.4,5 First, the sign of the imaginary part of the second order nonlinear susceptibility (Im χ(2)) measurable with HDVSFG provides information about the absolute (up/down) orientation of interfacial molecules.6 For instance, the sign of the OH vibrational stretch band of water at charged interfaces can indicate if the water molecules are, on average, pointed with their hydrogen atoms up or down at the interface.7,8 Second, and more importantly, the measurement of Im χ(2) allows one to obtain the “true” vibrational spectrum of the interface, which is free from interferences originating from the cross-terms © XXXX American Chemical Society
between nonresonant background and vibrational resonances. Therefore, the spectrum can be interpreted straightforwardly.9 In addition to the heterodyne detection, the use of isotopically diluted water, HOD-D2O, can “switch off” inter- and intramolecular vibrational couplings that complicate the spectral shape of H2O spectrum at the interfaces. We have used this strategy, i.e., combined use of HD-VSFG and isotopic dilution, to obtain vibrational spectrum of water at aqueous interfaces and acquired new insights.8−15 Previous reports have revealed how water molecules orient in the vicinity of charged interfaces (ionic surfactants or lipids present at the air/water interface). Water molecules are oriented with their hydrogen atoms pointed toward the interface (away from bulk; “H-up”) at the interface between water and negatively charged surfactant/lipid monolayers.7−9 In contrast, the orientation is opposite (“H-down”) when the charges on the surfactant or lipid headgroup are positive.7−9 These observations can be rationalized in terms of simple electrostatic charge-dipole interaction: The positive end of water dipole (H) is directed toward the interface in the case of a negatively charged interface, while the negative end (O) is directed toward the interface for positively charged interface. Received: September 6, 2016 Revised: September 22, 2016
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DOI: 10.1021/acs.jpcc.6b08980 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C
frequency: 2800−3700 cm−1, bandwidth: 300 cm−1, average power: 10 mW) beams are focused into Y-cut quartz (Crystal Base, Inc.; thickness: 10 μm) to generate local oscillator (LO). Transmitted ω1, ω2 and LO beams are refocused onto the sample surface with only LO being delayed by passing through a silica delay plate (thickness: 2 mm). The lipid monolayers were prepared on HOD in D2O subphase. The surface pressure was measured by a tension meter (Kibron Inc.) and kept in the range from 9 to 11 mN/m, which corresponds to the gel phase.28,29 HD-VSFG measurements were carried out for the ceramide/water interface, and the calibration for the phase and amplitude was made using z-cut quartz as the reference. The individual spectra measured with three distinct center frequencies were merged to obtain the full spectrum encompassing the entire OH stretch band of interfacial water with better signal-to-noise ratio.30 Materials. N-Stearoyl-D-erythro-sphingosine (C18-ceramide) was purchased from Avanti Polar Lipids, Inc., and dissolved in chloroform (min. 99%, Junsei Chemical Co., Ltd.) at the concentration of 1 mg/mL prior to the monolayer formation. Pure water (H2O) was obtained by Millipore purifier. D2O (99%, NMR grade) was purchased from Wako Chemicals. The composition of isotopes in HOD-D2O was H2O:HOD:D2O = 1:8:16. Molecular Dynamics Simulation. We carried out an allatom MD simulation of a hydrated C18-ceramide bilayer with explicit solvent molecules. The initial configuration was constructed using CHARMM-GUI Membrane Builder.31−33 The lipid bilayer that consists of 200 lipids (100 for each leaflet) was solvated in 150 mM KCl solution (8504 H2O and 32 KCl molecules). The modified version of CHARMM C36 force field25,34 and TIP3P model35 were employed for the ceramide and water, respectively. All bonds involving hydrogen atoms in lipids were constrained using SHAKE36 and water molecules were kept rigid using SETTLE.37 Long-range electrostatic interactions were evaluated using particle-mesh Ewald summation,38 while Lennard-Jones interactions were truncated at a cutoff distance of 12 Å with an atom-based force switching function that becomes effective at 10 Å. An equilibrium simulation was performed in a NPT ensemble (300 K, 1 bar). Langevin dynamics with a damping coefficient of 1 ps−1 was used for temperature control. A Langevin piston Nosé−Hoover method was used for pressure control with semi-isotropic pressure treatment, in which the pressure coupling is isotropic in the x and y directions but different in the z direction.39,40 A time step of 2 fs was used. The system was equilibrated for 20 ns, followed by a production run of 100 ns. The coordinates of hydrated ceramides were saved every 1 ps for the analysis. The calculated values of the area per lipid and bilayer thickness are 43.5 ± 0.4 Å2 and 44.8 ± 0.4 Å, respectively. These values agree reasonably with previously reported values for ceramides in a gel state.23,28,41−44 All simulations were performed using the NAMD program package.45
Water structure at zwitterionic lipid/water interface has been also studied for phosphatidylcholine (PC) monolayer.10 It was revealed that the overall orientation of the interfacial water exhibits a net H-up configuration although subensembles of Hup and H-down water molecules exist around in the vicinity of the negatively charged phosphate and positively charged choline moieties in the headgroup, respectively.10 Although the total charge of the lipid is neutral, the orientations of water molecules in the local environment of these two moieties are dictated by electrostatics and are opposite to each other. Because the effective charge experienced by the water molecules in the vicinity of the negatively charged phosphate group is greater than that around the positive choline group, the overall net orientation is observed to be H-up. The coexisting of H-up and H-down oriented water at the zwitterionic interface was also confirmed in the recent molecular dynamics (MD) simulation studies.16,17 In the case of the interfaces comprising lipids that are devoid of charged moieties, we have no a priori means for predicting the preferential orientation of interfacial water molecules. Therefore, it is intriguing to experimentally determine up/ down orientation of water at nonionic interface and to elucidate the origin of the preferential orientation. To achieve this, the combination of the HD-VSFG measurements and MD simulation is indispensable. In the present work, we study the N-stearoyl-D-erythrosphingosine (C18-ceramide)/water interface as a prototype of nonionic lipid (Scheme 1) interface by means of HD-VSFG Scheme 1. Molecular Structure of N-Stearoyl-D-erythrosphingosine (C18-Ceramide)a
a
The two hydroxyl groups discussed in this paper are indicated.
spectroscopy and MD simulation. The reason for choosing ceramide is twofold: First, ceramides constitute the backbone of all complex sphingolipids, such as sphingomyelins or glycosphingolipids. Since the properties of sphingolipids highly depend on the hydration level,18 it is important to elucidate water structure at the ceramide/water interface. Second, because of such biological interests, ceramides have been increasingly studied by MD simulations.18−24 Force field parameters for ceramide simulations have been actively developed23,25,26 and await experimental validation. The present HD-VSFG experiments reveal that ceramide interface exhibits a net H-up orientation of the water molecules. The corresponding MD simulation suggests that the presence of hydrogen (H)-bonding between the headgroup of ceramide and the water molecules in their vicinity as well as intralipid Hbonding is responsible for the H-up orientation.
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RESULTS AND DISCUSSION Figure 1 shows the complex χ(2) spectrum of the C18-ceramide monolayer/HOD-D2O interface in the frequency region between 2750 and 3800 cm−1. The Im χ(2) spectra show several sharp features below 3000 cm−1 and a broad feature above 3000 cm−1. Two negative sharp bands observed around 2870 and 2930 cm−1 are assigned to the CH3 symmetric stretching split by Fermi resonance with the bend overtone, and
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EXPERIMENTAL AND COMPUTATIONAL METHODS HD-VSFG Spectroscopy. The details of the experimental setup have been provided elsewhere.7,27 Briefly, the narrow band visible (ω1; center wavelength: 795 nm, bandwidth: 1.5 nm, average power: 10 mW) and broadband IR (ω2; center B
DOI: 10.1021/acs.jpcc.6b08980 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C
oxygen of the hydroxyl group can function as a H-bond acceptor from the adjacent interfacial water molecules that can induce H-up orientation. Nevertheless, the hydroxyl moiety may also act as a H-bond donor inducing H-down orientation, and hence it is not a trivial issue to anticipate the effect of hydroxyl moieties on the interfacial water orientation. In order to elucidate the origin of H-up orientation at the ceramide/water interface, MD simulation of a hydrated ceramide “bilayer” was carried out. The bilayer normal was chosen as the z-axis, and z = 0 was set at the bilayer center. We defined the interfacial water (|z| < 30 Å) according to the calculated electron density profile for various components (Figure 2a). This profile shows that water molecules penetrate well into the headgroup region including the amide and hydroxyl groups.
Figure 1. Imaginary (shaded red line) and real (black line) parts of χ(2) spectrum of the ceramide monolayer/HOD-D2O interface. The corresponding magnitude squared spectrum is also shown by blue line at the bottom.
the positive band at 2965 cm−1 is assigned to the CH3 antisymmetric stretching of the terminal methyl group.7,46 The negative sign for the symmetric stretch and positive sign for antisymmetric stretch indicate that the methyl group is oriented with their hydrogen pointing toward the air side.7,47,48 The symmetric and antisymmetric methyl stretch bands have different signs because of the opposite signs of the projections of the relevant molecular hyperpolarizability tensor components (βa,b,c) onto χ(2)yyz.48 The broad feature above 3000 cm−1 is due to OH stretch of water at the ceramide/water interface. The key observation is that the sign of the OH band is entirely positive. This indicates that the water molecules at ceramide monolayer interface are on average oriented with “H-up” orientation; i.e., their hydrogen atoms point away from bulk toward the lipid monolayer (“H-up” orientation).7 The OH band in the Im χ(2) spectrum appears as a single broad peak centered around 3400 cm−1. This indicates that the average Hbond strength at this interface is comparable to that in the bulk water.8,9 It should be noted that the positive OH band appearing in the Im χ(2) spectrum originates from the water molecules rather than the hydroxyl groups of the lipid. Since the transition dipole along the alcohol OH bond is expected to be directed from O atom to H atom, the alcohol OH band should appear with positive sign for H-up orientation or negative sign for Hdown orientation as in the case of water OH band.9,49 As verified by MD simulation, the alcohol OH is considered pointing down toward bulk water phase (average angle between O−H vector and z-axis is 122° and 139° for OH1 and OH3, respectively, in MD), so that the OH band due to alcohol OH should appear with a negative sign. The opposite is observed experimentally, indicating that the OH band arises not from the alcohol OH but from a different species, that is, the interfacial water molecules. Also, because water molecules in the vicinity of headgroups are much more numerous than ceramide’s hydroxyl groups, the χ(2) signal originating from the headgroups can be effectively swamped by the contributions from the interfacial water molecules. The preferred orientation of interfacial water cannot be dictated by electrostatics, since the headgroups of C18ceramide carries no charged moieties. It hints at the presence of other factors governing this preferred orientation. Intuitively, one might expect that the H-bonding between the interfacial water molecules and the hydroxyl moieties in the headgroup dictates the orientation of interfacial water molecules. The
Figure 2. (a) Electron density profile, (b) the population of H-up (Ppositive, cos θ > 0) and H-down (Pnegative, cos θ < 0) oriented water molecules in the vicinity of headgroup with different H-bond types (blue: donor (D) ∼ 9%, red: acceptor (A) ∼ 2%, green: in H-bond distance without H-bonding (I) ∼ 21%, gray: outside H-bond distance (O) ∼ 68%), and (c) the distribution of angle θ for the corresponding water molecules.
We analyzed the orientation of interfacial water by measuring the angle θ between the water dipole and bilayer normal. The value of θ is 90°) if water molecule is H-up (H-down) oriented. We divided water molecules into four types based on the H-bond status in order to relate their orientations and local interactions: water molecules donating H-bond to lipids (D), those accepting H-bond from lipids (A), those in the H-bond C
DOI: 10.1021/acs.jpcc.6b08980 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry C distance (3.5 Å) from lipid headgroup without forming H-bond with lipids (I), and those outside the H-bond distance from lipid headgroup (O). A geometrical threshold was used to define the H-bond, X−H···Y (X−Y distance
