Most naturally occurring metalloenzymes are gated by rate-limiting conformational changes and there exists a critical interplay between macroscopic structural changes and subatomic changes in electronic structure within embedded metallocofactors responsible for initiating catalysis. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we report conformationally switchable ArMs (swArMs) that are designed to undergo a large-scale conformational rearrangement upon effector binding at a site allosteric to the installed metallocofactor. Periplasmic binding proteins (PBPs) make an attractive host, as the conformations they undergo upon binding their substrates have been studied crystallographically. Using PBPs, various metallocofactors can be installed and the effect on reactivity studied as a function of conformational change. Our swArMs contain a Co(dmgH)2(X)(4-VP) cofactor (dmgH = dimethylglyoxime, 4-VP = 4-vinylpyridine, X = Cl–, N3–, H3C–) installed via cysteine ligation in the E. coli glutamine binding protein (GlnBP). We use energy dispersive X-ray fluorescence (ED-XRF) and Tandem Mass Spectrometry (MS) to show that the metallocofactor binds stoichiometrically and specifically to the protein host. Isothermal titration calorimetry (ITC) and fluorescence spectroscopy show that Gln binding is not affected by the presence of the metallocofactor. Initial studies reveal that in the case of [Co(dmgH)2(CH3)(4-VP)] attached to GlnBP, conformational change upon Gln binding triggers the Co-CH3 bond cleavage. The described research lays groundwork for studying how protein dynamics influence function, with the goal of understanding what makes native metalloproteins so special.

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

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