Phase Composition Control in Microsphere-Supported Biomembrane Systems

Abstract

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 (Ld) content. In addition, spectral imaging was used to assess the partitioning of FITC-labeled α-helical peptide between fluorescently labeled Ld phase and unlabeled ordered phase (Lo) phase lipid microdomains. The apparent peptide partition coefficient, Kp,app, was measured to be 0.89 ± 0.06, indicating a slight preference of the peptide for the Lo phase. A biomimetic motif of Lo phase concentration enhancement of 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 Ld and Lo phases, respectively.

Impact of poly(ethylene glycol) functionalized lipids on ordering and fluidity of colloid supported lipid bilayers

“…These form upon the dissolution of small unilamellar vesicles (SUVs), typically comprising zwitterionic phospholipids, into a dispersion of colloidal particles. [1][2][3][4][5][6][7][8][9][10] Interestingly, this strategy yields surfacefunctionalized colloids without direct chemical modification of the particle surface to introduce reactive groups. Instead, a SUV bilayer carrying membrane-bound additives is transferred onto the particle scaffold to confer attributes such as enhanced stability, fluorescence, and multivalent binding.…”

“…10,[13][14][15][16][17][18][19][20][21] The formation of CSLBs from the parent SUVs and colloidal particles, 5,13 such as silica, requires the lipid bilayer to be sufficiently fluid-like to enable the SUVs to rupture, spread and fuse to encapsulate the contacted particle. [1][2][3]18,[22][23][24][25][26] This is achieved at temperatures above the transition temperature, T m , of the constituent lipids, which depends markedly on lipid chemical structure, particularly on the tail length and carbon saturation. 27 In mixed membranes of immiscible lipids, the fluidity may further be exploited to generate spatiotemporal variations in composition due to phase separation.…”

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering

“…We conducted confocal-based fluorescence recovery after photobleaching (FRAP) studies to characterize the lateral mobility of the PEG-tethered PLB supported biomembranes. The effective diffusion coefficient of the embedded DiO reporter, D eff , can be determined as we have done in previous studies. − We also measured the average recovery fraction denoted as the mobile fraction, α. The results of FRAP measurements in the equatorial z section are tabulated in Figure .…”

“…Fluorescence recovery after photobleaching (FRAP) studies were carried out using the built-in protocol of the CLSM system, as we have implemented in multiple previous studies. − The image plane was set at the equator of the bead, and a 512 pixel × 32 pixel format was used (zoom value 16, scan speed 400 Hz, 488 nm AOTF 2% (power %)). This enabled the fast imaging (0.2 s/scan point) of two equatorially opposite ends of the bead.…”