Abstract
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.
Highlights
The classical textbook model of biomembrane structure, usually referred to as the fluid-mosaic model, envisions a twodimensional solution of integral membrane proteins in a homogeneous lipid solvent, albeit one composed of many molecular lipid species and possessing inside-outside asymmetry with respect to both protein and lipid components
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
DOPC/SM and POPC/SM binary mixtures yielded PWR spectra that can be ascribed to a superposition of two resonances corresponding to microdomains consisting of phosphatidylcholine- and SM-rich phases coexisting within a single bilayer
Summary
Lateral movement [14, 15]. Currently, the requirement for cholesterol in raft formation in biological membranes is unclear [13, 16, 17]. The most definitive studies have been done with model membranes (cf Ref. 2), with atomic force microscopy [13, 17] and fluorescence correlation spectroscopy [23] being effective in such systems These methodologies have clearly shown the spontaneous formation of laterally segregated domains within lipid bilayers and selective protein incorporation into such domains. We will demonstrate that plasmon-waveguide resonance (PWR) spectroscopy, applied to self-assembled solidsupported lipid bilayers, provides a uniquely useful method for observing raft formation in real-time and for obtaining information on the molecular composition of microdomains The uniqueness of this spectroscopic technique is that it allows the most important structural parameters of a lipid membrane, such as thickness, average surface area occupied by one lipid molecule (or molecular packing density), and degree of long-range molecular order, to be characterized for a single lipid bilayer in both steadystate and kinetic modes. We have incorporated a GPI-linked protein (placental alkaline phosphatase (PLAP)) into such bilayers and have shown that this protein preferentially associates with the SM-enriched microdomains
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