For the investigation of the function of membrane proteins and their interactions with cells, supported biomembrane systems are widely used in biomimetic materials. Our aim is to design alpha-helical peptide complexes to tether the membranes and enhance their stability and biological compatibility. In on-going work, we employ (K3A4L2A7L2A3K3) as anchoring molecules, where conjugation of the peptide with fluorescent probes at the orthogonal Lys(ivDde) terminal amino acid allows us to access a variety of chemistries (such as introducing fluorescent dye as probes, etc.) via orthogonal modification.
These peptides can grafted to surfaces using homobifunctional crosslinkers such as NHS-PEG3000-NHS to form tethering structures within proteolipobeads. We can control the site densities within supported bilayers by varying the mole fraction of peptides and lipids. Using flow cytometry, we determine the tethering site density of fluorophore-labled peptides. Moreover, the secondary structure of peptide within lipid structures is characterized using circular dichroism spectroscopy. Lateral fluidity of the fluorphore-tagged peptide is analyzed via fluorescence imaging microscopy (Confocal Microscopy) and quantified using fluorescence recovery after photobleaching (FRAP) techniques. Variations in the peptide sequence allow us to rationally investigate the influence of sequence on peptide anchor stability.
In our latest findings, the K3A4L2A7L2A3K3 peptide exhibits high alpha helical content for stable spanning within lipid bilayers. Furthermore, peptide anchors within tethered biomembranes with PEG3000 polymer cushions exhibits significant enhancements in mobility compared with untethered systems and give highly uniform surface coverage indicative of high quality supported membranes.