Payal_2026_Chembiochem_27_e70349

We previously introduced a genetically encoded, metal-responsive system for reversible control of protein function based on metal chelation by bipyridylalanine (BpyAla) residues. The efficacy of this linking group approach was demonstrated in two structurally and functionally distinct enzymes, Pyrococcus furiosus prolyl oligopeptidase (Pfu POP) and Photinus pyralis luciferase (Pluc). Here, we investigate the mechanistic basis of this switching in Pfu POP. Fluorescence-based metal competition assays and molecular dynamics (MD) simulations were conducted to quantify Ni(II) binding affinity and evaluate the structural response to Bpy(2)Ni(II) complex formation. (19)F NMR spectroscopy and MD simulations further indicate that linking group-controlled conformational changes near the catalytic triad, particularly within the loop containing H592, drive the observed activity modulation upon metal binding. These findings establish that genetically encoded metal-binding motifs can regulate enzyme function through subtle, localized conformational changes, providing a versatile platform for engineering responsive protein systems in synthetic biology, biosensing, and programmable catalysis.