Engineering a Conformationally Switchable Artificial Metalloprotein

Most naturally occurring metalloenzymes are gated by rate-limiting conformational changes and there exists a critical inter-play between macroscopic structural rearrangements of the protein, and subatomic changes affecting the electronic struc-ture of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structur-ally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conforma-tionally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime, X = N3–, H3C–, iPr–). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co‒S cysteine ligation within the E. coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction (2.99 Å resolu-tion) unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence and circular dichroism spectroscopies, along with isothermal titration calorimetry reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co‒S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.