Ribonucleotide reductases (RNRs) are ubiquitous and highly conserved enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides through a unique radical pathway. Because of their essential function, Class Ia RNRs, such as those in humans and E. coli, have been targeted in cancer therapeutics and are potential inhibitory targets for novel antibiotic compounds. Development of a molecule that stabilizes an inhibited quaternary structure is a logical strategy for Class Ia RNR inhibition. Currently, there are no facile assays for RNR quaternary structure. An ideal assay would be performed in the solution phase, spectroscopic, minimally structurally perturbative, and conducive to high throughput screening. We have developed a fluorescence anisotropy-based assay for changes in the quaternary structure of RNR, which is composed of α and β subunits whose stoichiometry is crucial in the regulation of activity. By labeling the β subunit with a long-lived transition metal luminophore, we are able to distinguish between the inhibited α4β4 and active α2β2 states through their unique anisotropy decays and estimate the rotational correlation times of each state. The observed rotational correlation times agree with steady-state anisotropy measurements. These results pave the way for the identification of inhibitors of E. coli RNR that function through stabilization of the α4β4 structure.

Developing time-resolved and steady-state fluorescence anisotropy assays to probe the quaternary structure of E. coli ribonucleotide reductase

ACS Spring 2022

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