Inhibitory Effect of Lanthanides on Native Lipid Flip-Flop

The influence of ytterbium ions (Yb³⁺), a commonly used paramagnetic NMR chemical shift reagent, on the physical properties and flip-flop kinetics of dipalmitoylphosphatidylcholine (DPPC) planar supported lipid bilayers (PSLBs) was investigated. Langmuir isotherm studies revealed that Yb³⁺ interacts strongly with the phosphate headgroup of DPPC, evidenced by the increases in shear and compression moduli. Using sum-frequency vibrational spectroscopy, changes in the acyl chain ordering and phase transition temperature were also observed, consistent with Yb³⁺ interacting with the phosphate headgroup of DPPC. The changes in the physical properties of the membrane were also observed to be concentration dependent, with more pronounced modification observed at low (50 μM) Yb³⁺ concentrations compared to 6.5 mM Tb³⁺, suggesting a cross-linking mechanism between adjacent DPPC lipids. Additionally, the changes in membrane packing and phase transition temperatures in the presence of Tris buffer suggested that a putative Yb(Tris)³⁺ complex forms that coordinates to the PC headgroup. The kinetics of DPPC flip-flop in the gel and liquid crystalline (lc) phases were substantially inhibited in the presence of Yb³⁺, regardless of the Yb³⁺ concentration. Analysis of the flip-flop kinetics under the framework of transition state theory revealed that the free energy barrier to flip-flop in both the gel and lc phases was substantial increased over a pure DPPC membrane. In the gel phase, the trend in the free energy barrier appeared to follow the trend in the shear moduli, suggesting that the Yb³⁺-DPPC headgroup interaction was driving the increase in the activation free energy barrier. In the lc phase, activation free energies of DPPC flip-flop in the presence of 50 μM or 6.5 mM Yb³⁺ were found to mirror the free energies of TEMPO-DPPC flip-flop, leading to the conclusion that the strong interaction between Yb³⁺ and the PC headgroup was essentially manifested as a headgroup charge modification. These studies illustrate that the presence of the lanthanide Yb³⁺ results in significant modification to the lipid membrane physical properties and, more importantly, results in a pronounced inhibition of native lipid flip-flop.