Tether-Supported Biomembranes with Alpha-Helical Peptide-Based Anchoring Constructs

The strict requirement of constructing a native lipid environment to preserve the structure and functionality of membrane proteins is the starting constraint when building biomaterials and sensor systems from these biomolecules. In order to enhance the viability of supported biomembranes anchored via polymer tethers, we have applied rationally designed peptide anchor technology to the lipid bilayer interface. For the investigation of the interactions between membrane proteins and lipid bilayers and the improvement of biomembrane-based materials, supported biomembrane systems are widely established and mimicked. Our aim is to design alpha-helical peptide complexes to tether the membranes and enhance their stability and biological compatibility. We employ (K 3A4L2A7L2A3K 3) as anchoring molecules, where conjugation of the peptide with fluorescein isothiocyanate (FITC) allows us to access a variety of chemistries (such as introducing fluorescent dye as probes, etc.) for orthogonal modification.;These peptides are designed to crosslink to amino-terminated surfaces and incorporated into DOPC lipid bilayers supported by microbeads. Here the silica bead (5microm) surface is biofunctionalized with NHS- PEG3000 -NHS as "polymer cushion" spacers. This is achieved by fusion of liposomes containing fluorescently labeled peptide and DOPC on silica microsphere surfaces. We aim to ultimately control the receptor site densities on lipobeads by varying the mole fraction of different lipids and ligands, determine the biomembrane distributions of fluorophore-labled lipid, ligand and receptors using flow cytometry and confocal microscopy. Moreover, hydrophobic peptide spanning within lipid bilayers is considered to mimic cytoskeletal stuctures in cellular biomembranes, and the helical structure of peptides within biomembranes is characterized with circular dichroism.;In the current study, we have designed peptide-based anchoring lipid bilayers that attach to a solid microparticle interface through integral tethering molecules (NHS-PEG3000-NHS). In earlier designs of tether-supported membranes, a wide variety of tethering molecules have been employed ranging from polymers to peptides. Thus far, primarily single lipid moieties connected at the end of tethers have been used to anchor membranes to solid supports. Our approach is to use an alpha helical peptide as a tethering molecule. Compared to a lipid tether and protein tether, which inserts into one leaflet of the lipid bilayer, peptide anchor is expected to impart more stability to the tethered bilayer. The control of variations in the peptide sequence and orthogonal modification of the fabricated supported biomembrane systems enables us to rationally investigate the influence the peptide structure in supported lipid bilayers.;Confocal microscopy was used to analyze the lipobead surface at different stages of surface modification by employing fluorophore staining techniques. Tether supported lipid bilayer membranes were constructed successfully on cushion tethered silica particles (5mum) using self-assembly method. Fluidity of the peptides witin supported membranes was quantified using fluorescence recovery after photobleaching (FRAP) technique.;Tether supported lipid bilayer membranes were constructed successfully on 5mum silica particles. The data on these systems show that the peptide exhibits a high alpha helical content when the peptide spans the lipid bilayers. Additionally, the diffusion coefficient of peptide anchors within polymer-cushioned and tethered biomembranes enhances peptide mobility compared with untethered systems. The results define a novel platform where the application of rationally designed peptide based anchors can be used to tether lipid bilayer, creating a more native lipid environment on spherical particles.