Ribonucleotide reductase of Escherichia coli. Cross-linking agents as probes of quaternary and quinary structure.

Abstract

The quaternary structure of ribonucleotide reductase of Escherichia coli was investigated, with the use of purified B1 and B2 proteins and bifunctional cross-linking agents. The holoenzyme is known to be an alpha 2 beta 2 tetramer consisting of two dimeric proteins: B1 (alpha 2) and B2 (beta 2). The cross-linking data support a model in which both of the beta subunits interact closely with only one of the two alpha subunits. Some of the interactions involving B2 were localized to the C terminus of the protein by use of truncated B2 protein (beta',beta'), a proteolytic cleavage product of B2 in which the 30 carboxy-terminal residues are missing from each of the beta subunits. Other interactions were indicated by the ability of glutaredoxin, but not thioredoxin, to inhibit some of the cross-linking reactions. We also asked whether ribonucleotide reductase interacts closely with other proteins inside the cell, by adding cross-linkers directly to suspensions of whole bacteria. Proteins in extracts of these cross-linked bacteria were resolved electrophoretically and probed with a monoclonal antibody to the B1 protein. High-molecular-mass products were detected, supporting the utility of this method for identifying intracellular interactions among enzymes of DNA precursor biosynthesis.