De novo design of Metalloproteins and metalloenzymes in a Three-Helix bundle

Abstract

For more than two decades, de novo protein design has proven to be an effective methodology for modeling native proteins. De novo design involves the construction of metal-binding sites within simple and/or unrelated α-helical peptide structures. The preparation of α 3 D, a single polypeptide that folds into a nativelike three-helix bundle structure, has signifi cantly expanded available de novo designed scaffolds. Devoid of a metal-binding site (MBS), we incorporated a 3Cys and 3His motif in α3 D to construct a heavy metal and a transition metal center, respectively. These efforts produced excellent functional models for native metalloproteins/metalloregulatory proteins and metalloenzymes. Morever, these α3 D derivatives serve as a foundation for constructing redox active sites with either the same (e.g., 4Cys) or mixed (e.g., 2HisCys) ligands, a feat that could be achieved in this preassembled framework. Here, we describe the process of constructing MBSs in α3 D and our expression techniques.