Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA binding protein

We show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable (nonequilibrium) conditions, reproducing a key feature of biological hard-tissue growth and assembly. The nonequilibrium synthesis of Cu₂O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein Tral in a room-temperature precursor electrolyte. The functional Tral derivative (TraIi1753::CN225) is engineered to possess a cysteine-constrained 12-residue Cu₂O binding sequence, designated CN225, that is inserted into a permissive site in TraI. When TraIi1753::CN225 is included in the precursor electrolyte, stable Cu ₂O nanoparticles form, even though the concentrations of [Cu ⁺] and [OH^-] are at 5% of the solubility product (K _sp,Cu2o). Negative control experiments verify that Cu₂O formation is controlled by inclusion of the CN225 binding sequence. Transmission electron microscopy and electron diffraction reveal a core-shell structure for the nonequilibrium nanoparticles: a 2 nm Cu₂O core is surrounded by an adsorbed protein shell. Quantitative protein adsorption studies show that the unexpected stability of Cu₂O is imparted by the nanomolar surface binding affinity of TraIi1753::CN225 for Cu₂O (K_d = 1.2 × 10^-8 M), which provides favorable interfacial energetics (-45 kJ/mol) for the core-shell configuration. The protein shell retains the DNA-binding traits of TraI, as evidenced by the spontaneous organization of nanoparticles onto circular double-stranded DNA.