Supramolecular Construction of Biohybrid Nanozymes Based on the Molecular Chaperone GroEL as a Promiscuous Scaffold

The study of enzymatic reactions in a confined space can provide valuable insight into the natural selection of nanocompartments for biocatalytic processes. Design of nanozyme capsules with the barrel-shaped protein cage of GroEL has been proposed as a promising means to constrain chemical reactions in a spatiotemporally controllable manner. Herein, we further demonstrate with hemin that the open GroEL cavity can provide a favorable microenvironment for shielding hydrophobic catalytically active species. Meanwhile, it is shown that the GroEL-caged hemin nanozyme not only has a significantly higher catalytic activity than merely dispersed hemin but also exhibits substrate specificity in the model oxidation reactions, which is a merit lacking in natural hemoproteins. To understand the underlying mechanism behind this supramolecular assembly, molecular docking and molecular dynamics simulations were performed to study the detailed interactions of hemin with the protein cage. This revealed the most likely binding mode and preferred binding residues in the paired hydrophobic α-helices lining the GroEL cavity which are genetically encoded for substrate capture. Finally, we demonstrate that the hemin-GroEL nanozyme has great potential in label-free fluorometric molecular detection when combined with suitable substrates such as homovanillic acid. We believe that our strategy is an advantageous tool for studying confined biocatalytic kinetics as simple mimics of protein-based organelles found in nature and for designing diverse nanozymes or bio-nanoreactors with the promiscuous GroEL binding cavity.