Tie-Line Determination by ESR for Liquid-Ordered and Liquid-Disordered Coexistence Regions in a Model Membrane

The phase hypothesis for “rafts” in the cell plasma membranes specifies that the lipid bilayer consists of two types of coexisting lamellar phases, the Lo phase containing a high amount of cholesterol and a Ld phase containing a lower amount of cholesterol. There are many well established methods to determine accurate phase boundaries of coexistence regions. However, determining the specific compositions of coexisting phase for lipid systems containing more than two lipid species remains very difficult. Methods have been proposed to determine one tie-line at a time, but not the entire field of tie-lines through all coexistence compositions, or the “global” tie-line configuration. The method we developed addresses this problem, and like the previous methods, is based on the linear superposition of magnetic resonance spectra. However, this method extends the analysis throughout the entire composition space of the coexistence region and is applied to find the best tie-line configuration for the Lo + Ld coexistence region of the “raft” model lipid mixture SPM/DOPC/cholesterol.

The essential aspects of the method are a well-determined phase boundary and a unique parameterization called a “ruled surface”. Least-squares fitting of the ESR data with the parameterized model result in a “global” tie-line configuration consistent with other known information of this lipid system. The best tie-line configuration is not parallel but exhibits a gradual change in slope with the smallest slope at the end tie-line of the neighboring three-phase region and the largest slope within the coexistence region connecting the boundary compositions with the highest and lowest cholesterol concentrations. An accurate determination of the “global” tie-line configuration of coexistence regions in model systems is a necessary step in understanding the lipid compositions of “rafts” in cell plasma membranes.

The lower left figure shows the variation of the ESR spectra around the two-phase boundary; the middle figure shows the compositions around and within the two-phase region that were studied; the figure on the right shows the set of tie-lines that were determined.