Probing Rigidity and Fluidity in the Interfacial Region of Lipid Bilayers with a Novel IR Probe

We report on development of a vibrational probe (N₃-C₁₂H₂₅, az12) suitable for reporting on the rigidity and fluidity of the interfacial region in lipid bilayers. Such probe can help assess critical biological functions of cell membranes, including membrane permeability. We demonstrated a high sensitivity of the az12 probe to the rigidity of the interfacial region, manifested in the sensitivity of the width of the N₃ moiety asymmetric stretching mode absorption peak. The structural and dynamic changes associated with the gel-liquid-crystal phase transition (Lβ-Lα) for three different lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and egg sphingomyelin (SM) were studied using FTIR and two-dimensional infrared (2DIR) spectroscopies. In addition to the new az12 probe targeting the interfacial region, the N₃-(CH₂)₁₁-CN probe (az11CN), recently developed to examine the hydrophobic region of bilayers, was also used, showing characteristic phase transitions for each bilayer at their characteristic phase transition temperatures. An order parameter, S_N3, corrected for the difference in the polarities of the interfacial and hydrophobic regions, was constructed. It shows how the overall rigidity of the bilayer is divided between the interfacial and hydrophobic regions, emphasizing their correlations. 2DIR spectral diffusion data were acquired for az12 and az11CN probes in the bilayer at various temperatures, reporting on inhomogeneous and homogeneous line width contributions (rigidity) and correlation times (fluidity) for the interfacial and hydrophobic regions. The spectral diffusion data for the interfacial region for all three bilayer types show large static inhomogeneous contributions, which are absent in the hydrophobic region data. The spectral diffusion data revealed differences for different bilayers, most apparent for the interfacial region. A greater increase in a homogeneous line width for SM with temperature, compared to that for DPPC and DPPG, could be linked to an increase in water permeability to the interfacial region of SM at higher temperatures. We found that the az12 probe constitutes a powerful reporter to measure rigidity (exerted angular constraints) and fluidity (time responses of the environment) in the interfacial region of a bilayer.