Methodology to probe subunit interactions in ribonucleotide reductases

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomelic precursors required for DNA replication and repair. Escherichia coli 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 Kd ≈ 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. © 2008 American Chemical Society.