Phospholipid vesicles exhibit a natural characteristic to fuse and reform into a continuous single bilayer membrane on hydrophilic solid substrates such as glass, mica, and silica. The resulting solid-supported bilayer mimics physiological tendencies such as lipid flip-flop and lateral mobility. The lateral mobility of fluorescently labeled lipids fused into solid-supported bilayers is found to change upon deposition on the membrane surface of an amphipathic alpha-helical peptide (AH) derived from the hepatitis C virus (HCV) NS5A protein. The binding of the AH peptide to a phospholipid bilayer, with the helical axis parallel to the bilayer, leads to immobilization of the bilayer. We used AFM to better understand the mechanistic details of this specific interaction, and determined that the diminished fluidity of the bilayer is due to membrane thinning. Utilizing this specific interaction between AH peptides and lipid molecules, we demonstrate a novel process for the creation of lipid partition by employing AH peptides as agents to immobilize lipid molecules, thus creating a patterned solid support with partition-defined areas of freely mobile lipid bilayers. This architecture could have a wide range of applications in novel sensing, biotechnology, high-throughput screening, and biomimetic strategies.

In this work, we studied the interactions of enzymes with model substrate surfaces using label-free techniques. Our model system was based on serine proteases (a class of enzymes that digests proteins) and surface-bound polypeptide substrates. While previous studies have focused on bulk media factors such as pH, ionic strength, and surfactants, this study focuses on the role of the surface-bound substrate itself. In particular, we assess how the substrate density of a polypeptide with an alpha-helical secondary structure influences surface reactivity. An alpha-helical secondary structure was chosen based on literature indicating that stable alpha-helices can resist enzymatic digestion. To investigate the protease resistance of a surface-bound a-helix, we designed an a-helical polypeptide (SS-polypeptide, where SS = disulfide), used it to form films of varying surface coverage and then measured responses of the films to enzymatic exposure. Using quartz-crystal microbalance with dissipation (QCM-D), angle-resolved X-ray photoelectron spectroscopy (AR-XPS), grazing-angle infrared spectroscopy (GAIRS), and other techniques, we characterized the degradation of films to determine how the lateral packing density of the surface-bound SS-polypeptide substrate affected surface proteolysis. Characterization of pure SS-polypeptide films indicated dense packing of helices that maintained their helical structure and were generally oriented normal to the surface. We found that films of pure SS-polypeptide significantly resisted enzymatic digestion, while incorporation of very minor amounts of a diluent in such films resulted in rapid digestion. In part, this may be due to the need for the enzyme to bind several peptides along the peptide substrate within the cleft for digestion to occur. Only SS-polypeptide films that were densely packed and did not permit catalytic access to multiple peptides (e.g., terminal peptides only) were resistant to enzymatic proteolysis.

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu2+ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu2+ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu2+ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu2+ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.