Most studies done to investigate biochemical reactions using artificial metalloproteins use rigid protein hosts and fail to consider the conformational dynamics of enzymes in living systems which affect the kinetics and thermodynamics of the catalyzed reactions. We have created switchable artificial metalloproteins (swArMs) that can undergo conformational changes upon effector binding at a site allosteric to the installed metallocofactor. Our approach combines the knowledge of the well-established ArMs, with conformational flexibility to link macroscopic structural changes with changes in the microenvironment around a metallocofactor. Periplasmic binding proteins (PBPs) present an excellent opportunity to function as hosts, as the conformations they undergo upon binding their substrates have been studied crystallographically. Various metallocofactors can be installed in PBPs and the effect on reactivity studied as a function of conformational change. Our swArMs contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime, X = N3–, H3C–, iPr–) installed via cysteine ligation in glutamine binding protein (GlnBP). We have used energy dispersive X-ray fluorescence (ED-XRF) and Mass Spectrometry (MS) to show that the metallocofactor binds stoichiometrically and specifically to the protein host via direct Co—S cysteine ligation. Isothermal titration calorimetry (ITC) and fluorescence spectroscopy show that Gln binding is not affected by the presence of the metallocofactor. Structural characterization by single-crystal X-ray diffraction (2.99 Å resolution) unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Kinetic studies reveal that in the swArMs containing a [Co(dmgH)2(CH3)] metallocofactor, conformational change upon Gln binding triggers the Co—S bond cleavage. The described research lays groundwork for studying how protein dynamics influence function and provides a platform for investigating the relationship between allostery and metallocofactor regulation.

Switchable artificial metalloproteins to probe conformational control in enzyme function

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