De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle.
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 α3D, a single polypeptide that folds into a native-like three-helix bundle structure, has significantly expanded available de novo designed scaffolds. Devoid of a metal-binding site (MBS), we incorporated a 3Cys and 3His motif in α3D 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 α3D 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 α3D and our expression techniques.
Department of Chemistry, University of Michigan, 930 North University Ave., Ann Arbor, MI, 48910, USA.
Jefferson S
JS
Plegaria
Department of Chemistry, University of Michigan, 930 North University Ave., Ann Arbor, MI, 48910, USA. vlpec@umich.edu.
Vincent L
VL
Pecoraro
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004798:Enzymes / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation
D004798:Enzymes; D008667:Metalloproteins
De novo protein design; Metal-binding site; Metalloenzyme; Metalloprotein; Metalloregulatory protein; Protein expression; Three-helix bundle