Design, engineering, and characterization of switchable artificial metalloproteins (swArMs)

Conformational dynamics are fundamental to metalloenzyme catalysis, yet engineering artificial systems that replicate these dynamics remains a significant challenge. This chapter discusses switchable artificial metalloproteins (swArMs), wherein synthetic metallocofactors are positioned within conformationally dynamic protein hosts. Focusing on protein hosts derived from periplasmic binding proteins (PBPs) such as the E. coli glutamine-binding protein (GlnBP), we outline strategies to select scaffolds with binary, ligand-driven structural changes, enabling precise control over the metallocofactor microenvironment. We detail key methodologies for site-specific metallocofactor incorporation, including cysteine mutagenesis and alkylation-based bioconjugation methods, emphasizing approaches to maintain conformational switching function. We further present comprehensive biophysical validation workflows, involving X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, isothermal calorimetry (ITC), and circular dichroism (CD) spectroscopy to correlate structural dynamics with changes in the metallocofactor environment. By integrating synthetic chemistry, biochemistry, and protein engineering, swArMs serve as a minimalist platform to investigate how conformational changes can modulate metalloenzyme catalysis in nature. These systems also show promise for applications in drug delivery, biocatalysis, and biosensing. Our goal is to provide researchers with a foundational toolkit for engineering dynamic metalloproteins, advancing both mechanistic understanding and practical innovations in bioinorganic chemistry.