Effect of Ca2+ to Sphingomyelin Investigated by Sum Frequency Generation Vibrational Spectroscopy  Rong-juan Feng, Lu Lin, Yi-yi Li, Ming-hua Liu, Yuan Guo, Zhen Zhang  Biophysical Journal  Volume 112, Issue 10, Pages 2173-2183 (May 2017) DOI: 10.1016/j.bpj.2017.04.026 Copyright © 2017 Biophysical Society Terms and Conditions

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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Given here are the surface pressure (mN/m)-area (Å2) isotherms 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 Ca2+ ions into the subphase, the ESM isotherm shifts to higher MMA, indicating that Ca2+ 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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Shown here are HR-BB-SFG-VS spectra of ESM monolayer films on the pure water and CaCl2 solution subphases at (a) 68 Å2/molecule and (b) 52 Å2/molecule under ssp and ppp polarization. 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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions

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) the 68 Å2/molecule (solid lines are fitting results using Lorentzian lineshape functions, the fitting parameters are listed in Table S2) and (b) the 52 Å2/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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions

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 with (a) 68 and (b) 52 Å2/molecule in the C–H stretching region. The overall 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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions

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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 Given here is the molecular mechanism of Ca2+ ions interacting with the headgroup and hydrocarbon chains of ESM. After adding Ca2+ 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. Biophysical Journal 2017 112, 2173-2183DOI: (10.1016/j.bpj.2017.04.026) Copyright © 2017 Biophysical Society Terms and Conditions