Engineering the metal-binding loop at a type 1 copper center by circular permutation

Cupredoxins are electron transfer proteins with a single type 1 copper center. A metal-binding loop in the protein harbors 3 of the copper ligands. Previous research has altered the sequence and length of the loop, changing the spectroscopic properties and reduction potentials of cupredoxins. It remains elusive whether the loop is flexible enough to tolerate long sequences. In this work we extended the loop to infinity by performing circular permutation on azurin, a typical cupredoxin. The mutant protein, Az-loop was characterized by absorption spectroscopy, electron paramagnetic resonance spectroscopy, cyclic voltammetry and X-ray crystallography. The results further confirm the flexibility of the metal-binding loop.