1H NMR studies of mouse ribonucleotide reductase: the R2 protein carboxyl-terminal tail, essential for subunit interaction, is highly flexible but becomes rigid in the presence of protein R1.

Mouse ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. It has earlier been shown that the carboxyl-terminal part of the R2 protein is essential for subunit association to form the active enzyme complex. We now demonstrate that protein R2 gives rise to a number of sharp 1H NMR resonances, significantly narrower than the major part of the resonances. This line narrowing of certain resonances indicates segmental mobility in the molecule. In two-dimensional 1H TOCSY spectra of protein R2, cross-peak patterns from about 25 amino acid residues are visible. Most of these were assigned to the carboxyl-terminal part of the protein by comparisons with cross-peak patterns of oligopeptides corresponding to the carboxyl terminus of mouse R2 and to the patterns of a seven amino acid residue carboxyl-terminal truncated form of protein R2. These results and the magnitude of the chemical shifts of the assigned residues demonstrate that the carboxyl-terminal part of mouse R2 protein is highly mobile compared to the rest of the protein and essentially unstructured. When protein R1 is added to a solution of protein R2, the sharp resonances are broadened, suggesting that the mobility of the carboxyl-terminal tail of protein R2 is reduced. The possibility of making direct observations of subunit interaction in native and mutagenized R1/R2 proteins should allow discrimination between effects of amino acid replacements on the catalytic mechanism and effects on subunit interaction.