Engineering a Conformationally Switchable Artificial Metalloprotein

• DOI: 10.1021/jacs.2c08885
• Publication Date: 2022-11-15T16:38:33Z
• Authors: Saman Fatima; David G. Boggs; Noor Ali; Peter J. Thompson; Megan C. Thielges; Jennifer Bridwell...; Lisa Olshansky
• Keywords: Isothermal Titration Calorimetry; Conformational change; Metalloprotein

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