Mapping protein-protein interactions by localized oxidation: consequences of the reach of hydroxyl radical

Biochemistry. 2009 Jun 2;48(21):4577-86. doi: 10.1021/bi900273j.


Hydroxyl radicals generated from a variety of methods, including not only synchrotron radiation but also Fenton reactions involving chelated iron, have become an accepted macromolecular footprinting tool. Hydroxyl radicals react with proteins via multiple mechanisms that lead to both polypeptide backbone cleavage events and side chain modifications (e.g., hydroxylation and carbonyl formation). The use of site-specifically tethered iron chelates can reveal protein-protein interactions, but the interpretation of such experiments will be strengthened by improving our understanding of how hydroxyl radicals produced at a point on a protein react with other protein sites. We have developed methods for monitoring carbonyl formation on proteins as a function of distance from a hydroxyl generator, iron-(S)-1-[p-(bromoacetamido)benzyl]EDTA (FeBABE), conjugated to an engineered cysteine residue. After activation of the chelated iron with ascorbate and peroxide produces new protein carbonyl groups, their positions can be identified using element-coded affinity tagging (ECAT), with carbonyl-specific tags {e.g., rare earth chelates of (S)-2-[4-(2-aminooxy)acetamidobenzyl]-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (AOD)} that allow for affinity purification, identification, and relative quantitation of oxidation sites using mass spectrometry. Intraprotein oxidation of single-cysteine mutants of Escherichia coli sigma(70) by tethered FeBABE was used to calibrate the reach of hydroxyl radical by comparison to the crystal structure; the application to protein-protein interactions was demonstrated using the same sigma(70) FeBABE conjugates in complexes with the RNA polymerase core enzyme. The results provide fundamental information for interpreting protein footprinting experiments in other systems.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cattle
  • Cysteine
  • DNA-Directed RNA Polymerases / chemistry
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Edetic Acid / analogs & derivatives
  • Edetic Acid / metabolism
  • Escherichia coli / enzymology
  • Hydroxyl Radical / metabolism*
  • Models, Molecular
  • Mutation
  • Oxidation-Reduction
  • Protein Binding
  • Protein Conformation
  • Thermus / enzymology


  • Hydroxyl Radical
  • 1-(4-bromoacetamidobenzyl)EDTA
  • Edetic Acid
  • DNA-Directed RNA Polymerases
  • Cysteine