Methodology to probe subunit interactions in ribonucleotide reductases.
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.
Hassan
A Quamrul
AQ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Wang
Yongting
Y
Plate
Lars
L
Stubbe
JoAnne
J
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
D001577:Benzophenones; D003432:Cross-Linking Reagents; D021122:Protein Subunits; D015081:2-Naphthylamine; D012264:Ribonucleotide Reductases; D003545:Cysteine