Probing the Hydrophobic Region of a Lipid Bilayer at Specific Depths Using Vibrational Spectroscopy

A novel spectroscopic approach for studying the flexibility and mobility in the hydrophobic interior of lipid bilayers at specific depths is proposed. A set of test compounds featuring an azido moiety and a cyano or carboxylic acid moiety, connected by an alkyl chain of different lengths, was synthesized. FTIR data and molecular dynamics calculations indicated that the test compounds in a bilayer are oriented so that the cyano or carboxylic acid moiety is located in the lipid head-group region, while the azido group stays inside the bilayer at the depth determined by its alkyl chain length. We found that the asymmetric stretching mode of the azido group (ν_N3) can serve as a reporter of the membrane interior dynamics. FTIR and two-dimensional infrared (2DIR) studies were performed at different temperatures, ranging from 22 to 45 °C, covering the Lβ-Lα phase transition temperature of dipalmitoylphosphatidylcholine (∼41 °C). The width of the ν_N3 peak was found to be very sensitive to the phase transition and to the temperature in general. We introduced an order parameter, S_N3, which characterizes restrictions to motion inside the bilayer. 2DIR spectra of ν_N3 showed different extents of inhomogeneity at different depths in the bilayer, with the smallest inhomogeneity in the middle of the leaflet. The spectral diffusion dynamics of the N₃ peak was found to be dependent on the depth of the N₃ group location in the bilayer. The obtained results enhance our understanding of the bilayer dynamics and can be extended to investigate membranes with more complex compositions.