pH-Responsive HierarchicalSelf-Assembly of EngineeredProtein–Gold Nanocluster Conjugates with Tunable Fluorescence

Abstract We report the rational engineering of a two-α-helix protein via site-directed mutagenesis, wherein histidine residues at the native catalytic active site were substituted with cysteines and tyrosine residues were introduced in close proximity to these cysteine residues. This modification enabled the site-directed reduction and nucleation of free gold ions into size-tunable gold nanoclusters (AuNCs) exhibiting red, green, and blue photoluminescence upon ultraviolet excitation. Furthermore, by modulating the solution pH, we successfully fabricated nanofibers via the co-assembly of the protein and AuNCs. Comprehensive structural characterization confirmed the architecture of these biohybrid materials. Protein-AuNCs can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), exhibiting peroxidase-like activity under specific conditions. Furthermore, this peroxidase-like activity was successfully applied to the colorimetric detection of glutathione with a linear response in the concentration range of 100–300 μM.