The methodology for constructing recombinant proteins, whose original functions can be manipulated with desirable external stimuli such as ions, pH, photo-irradiation? or ligand binding, is an attractive target in protein design. In particular, applying them as functional macromolecule, such as sensor proteins, ligand-dependent enzymes (in vivo or in vitro), tuning their functions possible with the external specific ligand is quite versatile. So far we studied the design of de novo coiled-coil proteins, whose tertiary structures can be significantly changed from a random coil to the folded coiled-coil structure, with response to the external stimuli (pH change, binding of metal ion or peptide ligand).
With the insertion of such a metamorphosis protein sequence into the scaffolds of target natural proteins, we succeeded to construct recombinant proteins, having each desirable ligand-dependency, such as the ligand-dependent RNaseT1, metal-ion-dependent green fluorescent protein (GFP), and ligand-dependent T7 RNAP/T7 lysozyme complex.