Phase Composition Control in Microsphere-Supported Biomembrane Systems

The popularization of studies in membrane protein lipid phase coexistence has prompted the development of new techniques to construct and study biomimetic systems with cholesterol-rich lipid microdomains. Here, microsphere-supported biomembranes with integrated α-helical peptides, referred to as proteolipobeads (PLBs), were used to model peptide/protein partitioning within DOPC/DPPC/cholesterol phase-separated membranes. Due to the appearance of compositional heterogeneity and impurities in the formation of model PLB assemblies, fluorescence-activated cell sorting (FACS) was used to characterize and sort PLB populations on the basis of disordered phase (L_d) content. In addition, spectral imaging was used to assess the partitioning of FITC-labeled α-helical peptide between fluorescently labeled L_d phase and unlabeled ordered phase (L_o) phase lipid microdomains. The apparent peptide partition coefficient, K_p,app, was measured to be 0.89 ± 0.06, indicating a slight preference of the peptide for the L_o phase. A biomimetic motif of the L_o phase concentration enhancement of the biotinyl-peptide ligand display in proteolipobeads was also observed. Finally, peptide mobility was measured by FRAP separately in each lipid phase, yielding diffusivities of 0.036 ± 0.005 and 0.014 ± 0.003 μm²/s in the L_d and L_o phases, respectively.