Cell-Free Protein Synthesis of Metalloproteins.

Metalloproteins, proteins containing metal atoms or clusters within their structures, are critical for various biological functions across all domains of life. More than hundreds of different types have been discovered, which conduct various roles such as transportation of O₂, catalyzing chemical reactions, sensing environmental changes, and relaying electrons. Metalloprotein molecules incorporate a variety of metal atoms, coordinated to specific amino acid residues that affect their conformation and functionality. The process of metal incorporation typically occurs during or post-protein folding, often requiring chaperones for metal ion delivery and quality control. Progress in understanding metal incorporation and metalloprotein functionality has been enhanced by cell-free protein synthesis (CFPS) methods that offer direct control over the synthesis environment. This chapter reviews the diverse applications of CFPS methods in metalloprotein research, encompassing structure-function studies, protein engineering, and creation of artificial metalloproteins. Examples demonstrating the utility and advances brought about by CFPS in synthetic biology, electrochemistry, and drug discovery are highlighted. Despite remarkable progress, challenges remain in optimizing and advancing the CFPS methods, underscoring the need for future explorations in this transformative approach to metalloprotein study and engineering.