Methodology To Probe Subunit Interactions in Ribonucleotide Reductases

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. <i>Escherichia coli</i> RNR is a 1:1 complex of two homodimeric subunits, α2 and β2. The interactions between α2 and β2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356−375) of β2. To study subunit interactions, a single reactive cysteine has been introduced into each of 15 positions along the C-terminal tail of β2. Each cysteine has been modified with the photo-cross-linker benzophenone (BP) and the environmentally sensitive fluorophore dimethylaminonaphthalene (DAN). Each construct has been purified to homogeneity and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) and electrospray ionization mass spectrometry (ESI-MS). Each BP-β2 has been incubated with 1 equiv of α2 and photolyzed, and the results have been analyzed quantitatively by SDS−PAGE. Each DAN-β2 was incubated with a 50-fold excess of α2, and the emission maximum and intensity were measured. A comparison of the results from the two sets of probes reveals that sites with the most extensive cross-linking are also associated with the greatest changes in fluorescence. Titration of four different DAN-β2 variants (351, 356, 365, and 367) with α2 gave a <i>K</i>_d ≈ 0.4 μM for subunit interaction. Disruption of the interaction of the α2–DAN-β2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high-throughput screen for inhibitors of subunit interactions