H-1-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 tha t the carboxyl-terminal part of the R2 protein is essential for subuni t association to form the active enzyme complex. We now demonstrate th at protein R2 gives rise to a number of sharp H-1 NMR resonances, sign ificantly narrower than the major part of the resonances. This line na rrowing of certain resonances indicates segmental mobility in the mole cule. In two-dimensional H-1 TOCSY spectra of protein R2, cross-peak p atterns from about 25 amino acid residues are visible. Most of these w ere assigned to the carboxyl-terminal part of the protein by compariso ns with cross-peak patterns of oligopeptides corresponding to the carb oxyl terminus of mouse R2 and to the patterns of a seven amino acid re sidue carboxyl-terminal truncated form of protein R2. These results an d the magnitude of the chemical shifts of the assigned residues demons trate that the carboxyl-terminal part of mouse R2 protein is highly mo bile compared to the rest of the protein and essentially unstructured. When protein R1 is added to a solution of protein R2, the sharp reson ances are broadened, suggesting that the mobility of the carboxyl-term inal tail of protein R2 is reduced. The possibility of making direct o bservations of subunit interaction in native and mutagenized R1/R2 pro teins should allow discrimination between effects of amino acid replac ements on the catalytic mechanism and effects on subunit interaction.