Designing functional nanomaterials from stable protein 1

Abstract

Proteins are utilised as building blocks, or tectons, in bionanotechnology, for simple and cost-effective synthesis of complex nanostructures. Ideally, these tectons should form controllable, ordered arrays, which are well defined and stable to different conditions encountered during synthesis and application. Stable protein 1 (SP1) is an exceptionally stable nanoring, which is resistant to a range of different conditions, making it an ideal bionanotechnology tecton. This study developed novel SP1 tectons with the incorporation of an unnatural amino acid, p-azido-L-phenylalanine at two geometrical orientations in the nanoring (lateral and vertical tectons). Following click chemistry for site-specific biorthogonal conjugation, a range of novel functionalised SP1 nanomaterials were produced. Bacterial cellular machinery was exploited to co-translationally encode p-azido-L-phenylalanine using the amber codon suppression technique. Strain-promoted azide-alkyne cycloaddition was established at the azide sites,simply by mixing and stirring with alkyne-functionalised molecules. Subsequently, a range offunctional particles (fluorophores, gold nanoparticles, and enzymes) were selectively attachedonto the SP1 scaffolds. Mass spectrometry confirmed the incorporation of p-azido-L-phenylalanine and calculated at greater than 95% efficiency for both mutants. Strain-promotedazide-alkyne cycloaddition of the florescent dye, Cyanine 5, and subsequent fluorescenceimaging further confirmed the incorporation. Size exclusion chromatography and transmissionelectron microscopy also revealed successful conjugation of green fluorescent protein, glucoseoxidase and, horseradish peroxidase, and gold nanoparticles with both SP1 tectons, and thescaffolding of two redox proteins forming an enzymatic cascade. This study introduces newSP1 tectons as robust scaffolds to build functional bionanohybrids that may form componentsof future nanodevices.