Creation and Relaxation of Phospholipid Compositional Asymmetry in Lipid Bilayers Examined by Sum-Frequency Vibrational Spectroscopy

Eukaryotic cells contain an asymmetric distribution of phospholipids in the two leaflets of the lipid bilayer which is known to contribute to cellular function.(1-12) In the plasma membrane of eukaryotic cells, the aminophospholipids with phosphatidylserine (PS) and phosphatidylethanolamine (PE) headgroups are predominately located on the cytosolic leaflet while sphingolipids with phosphatidylcholine (PC) headgroups and sphingomeylin are on the extra-cellular leaflet.(1-11) There have been a number of theories about the mechanism of transbilayer movement of lipids in cellular systems and the physical process by which lipid compositional asymmetry in the plasma membrane of eukaryotic cells is maintained. It is generally accepted that a significant barrier to native lipid translocation (movement between leaflets of the bilayer) exists which is related to the energetic penalty of moving the hydrophilic headgroup of a phospholipid through the hydrophobic core of the membrane.(13,14) Overcoming this energetic barrier represents the rate limiting step in the spontaneous flip-flop of phospholipids in biological membranes, yet, while numerous kinetic studies of phospholipid flip-flop have been conducted(15-22), few researchers have reported thermodynamic parameters for the process.(19-21) Using methods of classical surface chemistry coupled with nonlinear optical methods, we have developed a novel analytical approach, using sum-frequency vibrational spectroscopy (SFVS), to selectively probe lipid compositional asymmetry in a planar supported lipid bilayer. This new method allows for the detection of lipid flip-flop kinetics and compositional asymmetry without the need for a fluorescent or spin-labeled lipid species by exploiting the coherent nature of SFVS. The SFVS intensity arising from the terminal methyl groups of the lipid fatty acid chains is used as an internal probe to directly monitor the compositional asymmetry in planar supported lipid bilayers (PSLBs). By selectively deuterating a sub-population of lipids, the SFVS intensity is proportional to the population difference between hydrogenated lipids in the top, N(T), and the bottom, N(B), leaflets due to the cancellation of the SFVS signal arising from lipids hydrogenated residing in an anti-parallel arrangement, (17,23-26) allowing us to directly relate the measured intensity to the population difference in the bilayer (Equation 1) and provides a direct measure of the percent asymmetry (%AS) in the membrane (Equation 2). In this presentation, the effect of lipid composition, headgroup and fatty acid chemical structure, on the rate and thermodynamics of lipid transbilayer migration and the electrostatic induction of lipid asymmetry will be discussed.