Effects of Polyunsaturated Acyl Chains on Coexisting Liquid Ordered/Liquid Disordered Phases

Ternary mixtures of POPC, sphingomyelin (SM) and cholesterol (Chol) form coexisting liquid ordered and liquid disordered fluid phases over a wide range of molar ratios. We examined the effects of polyunsaturated acyl chains in this ternary system by incrementally exchanging SDPC (18:0,22:6 PC) for POPC. All measurements were performed on extruded LUVs formed above the main phase transition of the SM. Changes in bilayer properties of the two phases were monitored with NBD linked to di-16:0 PE, which partitions into the liquid ordered phase, and NBD linked to di-18:1 PE which partitions into the liquid disordered phase. Fluorescence lifetime and anisotropy decay dynamics of both fluorescent probes were assessed via frequency-domain measurements collected from 5 to 300 MHz. In both phases the exchange of SDPC for POPC reduced the average fluorescence lifetime of the two probes from the value observed in 1/1/1 POPC/SM/chol. In both phases the lifetime was reduced to a value similar to that observed in a ternary mixture consisting of 8/1/1 POPC/SM/chol. In the liquid disordered phase the addition of SDPC had essentially no influence on probe dynamics, while in the liquid ordered phase SDPC caused a significant acceleration of probe rotational motion. This result was unexpected since it was assumed that the SDPC would be present only in the liquid disordered phase. FRET measurements using di-16:0-PE labeled with NBD and di-18:1 PE labeled with rhodamine were performed in order to monitor changes possible changes in domain size induced by the exchange of POPC for SDPC. Initial results show similar transfer efficiency between these two probes in 1/1/1 POPC/SM/chol and in 1/2/3/3 POPC/SDPC/SM/chol, suggesting the presence of SDPC does not greatly perturb domain size and structure.