(114c) Incorporation and Characterization of Alpha-Helical Peptide-Based Anchors for Bead-Supported Lipid Bilayers

Our aim is to design alpha-helical peptide anchors to enhance the stability and biological relevance of lipid bilayers for the study of membrane proteins. We employ (K3A4L2A7L2A3K3) as anchoring molecules, where conjugation of the peptide with N-hydroxysuccinimide (NHS)-esters and SATP (N-succinimidyl S-acetylthiopropionate) through amine-based coupling allows us to covalently graft beads with our peptide in a quantifiable fashion. These peptides are also conjugated to NHS-PEG-maleimide, subsequently used to anchor lipid bilayers to the microbead surface. Several experiments are used to quantify the surface coverage of peptide and lipids. We control the membrane protein densities on lipobeads by varying the mole fractions of proteoliposomes. Using flow cytometry, we determine the surface density of fluorophore-labeled lipids and target membrane proteins. Lateral fluidity of the fluorescently-tagged peptides and lipids are analyzed via confocal fluorescence imaging microscopy and quantified using fluorescence recovery after photobleaching (FRAP). Moreover, the secondary structure of peptides within the bilayer is characterized with circular dichroism. Lastly, we inspect the peptide-based anchor in detail via Nuclear Magnetic Resonance (NMR), which will supply data to evaluate the structural properties of the anchor within the bilayer and in detergent micelles. Variations in the peptide sequence allow us to rationally investigate the influence of peptide sequence on bead-supported biomembrane stability.