Immobilization and enhancement of a heterodimeric fluorescence biosensor in fibrous protein biomaterials

Leave-one-out green fluorescent proteins (LOO_GFPs) have a reduced quantum yield relative to the parent protein and form fluorescent oligomers in the unbound state. Immobilizing LOO_GFPs in materials composed of the Drosophila protein Ultrabithorax (Ubx) via gene fusion increased the fluorescent signal, significantly stabilized the biosensor, and prevented oligomerization into fluorescent aggregates, which has the potential to elevate the sensor's noise well above the signal. Interactions between LOO_GFP and Ubx hampered analyte rebinding. By optimizing the concentrations of LOO_GFP, salt, and detergent in the assay, the signal to noise ratio for the biosensor increased fourfold. These modified fibers represent the first incorporation of a protein complementation assay into protein-based materials, as well as the first incorporation, via gene fusion, of a heterodimeric functional protein into materials composed of a different self-assembling protein. This study highlights the advantages and identifies potential pitfalls associated with protein immobilization in materials.