Streptavidin-based nanostructures : from bulk to surface-confined assembly studies

Self-assembly, the ability of disordered units to spontaneously organize themselves into high-order structures via non covalent bonds, is a physical principle allowing nanostructures to be created from the bottom-up, extending nano-order to macroscales. This thesis was dedicated to program the self-organization of mature wild-type streptavidin by using linear tetrabiotinylated and trifurcated hexabiotinylated connectors. Self-organization studies in bulk solution demonstrated that streptavidin combined with a linear tetrabiotinylated connector spontaneously assembled into a one-dimensional streptavidin-based block copolymer. In the presence of calcium ions, the fibrils formed bundles that served as a template for the nucleation, the growth and the assembling of calcite microcrystals. This hierarchical self-assembly process yielded millimeter-sized one-dimensional mineralized protein matrices. Both mineralized and naked one-dimensional streptavidin-based block co- polymers were imaged by electron microscopy. With the aim of obtaining well-defined and monodisperse nanostructures, a step-by- step assembly approach was investigated. Designed mixed bis-biotinylated monolayers on gold were used to anchor streptavidin, which was successively exposed to linear tetrabiotinylated or trifurcated hexabiotinylated connectors and streptavidin. The stepwise elongation process was followed in situ by surface plasmon resonance and quartz crystal microbalance with dissipation monitoring. The size-controlled immobilized streptavidin-based fibrils were scrutinized by atomic force microscopy in the hydrated state

Chemically Programmed Supramolecular Assembly of Hemoprotein and Streptavidin with Alternating Alignment

It is shown that an intramolecularly linked cofactor dyad enforces the assocn. of two different proteins according to a predefined program.  The present study demonstrates that the heme-bis(biotin) conjugate contains the information to produce a stable 2:1 Mb-SAv complex.  Propagation of this moiety into a polymer results from using a dimericMb building block.  The supramol. composite fiber obsd. by AFM techniques is the first example of a chem. programmed heterotropic protein copolymer with alternating alignment.  The heme cofactor's dioxygen binding function is maintained upon incorporation within the fiber, suggesting that this approach is well-suited for the creation of functional nanobiomaterials.  Immobilization of the first building block will allow to: (1) study the programmed assembly by quartz microbalance anal., (2) increase the complexity by varying the capping groups on the dyad and/or (3) introduce addnl. protein building blocks to afford protein polymers with a desired sequence and function.  Potential applications include multifunctional catalysis, artificial photosynthesis, and smart nanomaterials for medical applications

Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer

Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His(6)-tagged b-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His(6)-BACE in the Ni2+-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response

A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection

This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4+ T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4+ T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza