title: 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. Escherichia coli RNR is a 1:1 complex of two homodimeric subunits, alpha2 and beta2. The interactions between alpha2 and beta2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356-375) of beta2. To study subunit interactions, a single reactive cysteine has been introduced into each of 15 positions along the C-terminal tail of beta2. 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-beta2 has been incubated with 1 equiv of alpha2 and photolyzed, and the results have been analyzed quantitatively by SDS-PAGE. Each DAN-beta2 was incubated with a 50-fold excess of alpha2, 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-beta2 variants (351, 356, 365, and 367) with alpha2 gave a K(d) approximately 0.4 microM for subunit interaction. Disruption of the interaction of the alpha2-DAN-beta2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high-throughput screen for inhibitors of subunit interactions.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: A Quamrul
authors.initials: AQ
authors.lastname: Hassan
authors.forename: Yongting
authors.initials: Y
authors.lastname: Wang
authors.forename: Lars
authors.initials: L
authors.lastname: Plate
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D015081:2-Naphthylamine / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry; D001709:Biotechnology; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D001577:Benzophenones; D003432:Cross-Linking Reagents; D021122:Protein Subunits; D015081:2-Naphthylamine; D012264:Ribonucleotide Reductases; D003545:Cysteine