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

The interactions between Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ ions can induce ordering of the acyl chains in the ESM monolayer. An analysis of the one alkyl-chain-deuterated ESM revealed that the Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 lipid-cation 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.