Plasmon-waveguide resonance studies of lateral segregation of lipids and proteins into microdomains (rafts) in solid-supported bilayers

Plasmon- waveguide resonance ( PWR) spectroscopy has been used to examine solid- supported lipid bilayers consisting of dioleoylphosphatidylcholine ( DOPC), palmitoyloleoylphosphatidylcholine ( POPC), sphingomyelin ( SM), and phosphatidylcholine/ SM binary mixtures. Spectral simulation of the resonance curves demonstrated an increase in bilayer thickness, long- range order, and molecular packing density in going from DOPC to POPC to SM single component bilayers, as expected based on the decreasing level of unsaturation in the fatty acyl chains. DOPC/ SM and POPC/ SM binary mixtures yielded PWR spectra that can be ascribed to a superposition of two resonances corresponding to microdomains ( rafts) consisting of phosphatidylcholine- and SM- rich phases coexisting within a single bilayer. These were formed spontaneously over time as a consequence of lateral phase separation. Each microdomain contained a small proportion (< 20%) of the other lipid component, which increased their kinetic and thermodynamic stability. Incorporation of a glycosylphosphatidylinositol- linked protein ( placental alkaline phosphatase) occurred within each of the single component bilayers, although the insertion was less efficient into the DOPC bilayer. Incorporation of placental alkaline phosphatase into a DOPC/ SM binary bilayer occurred with preferential insertion into the SM- rich phase, although the protein incorporated into both phases at higher concentrations. These results demonstrate the utility of PWR spectroscopy to provide insights into raft formation and protein sorting in model lipid membranes.