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

Sarah M. Cheal, Mindy Ng, Brianda Barrios, Zheng Miao, Amir K. Kalani, Claude F. Meares

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 sitespecifically 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 σ70 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 σ70 FeBABE conjugates in complexes with the RNA polymerase core enzyme. The results provide fundamental information for interpreting protein footprinting experiments in other systems.

Original languageEnglish (US)
Pages (from-to)4577-4586
Number of pages10
JournalBiochemistry
Volume48
Issue number21
DOIs
StatePublished - Jun 2 2009

Fingerprint

Hydroxyl Radical
Oxidation
Proteins
Cysteine
Protein Footprinting
Iron
Protein Carbonylation
Iron Chelating Agents
Synchrotrons
Peroxides
DNA-Directed RNA Polymerases
Hydroxylation
Mass Spectrometry
Synchrotron radiation
Escherichia coli
Rare earths
Purification
Mass spectrometry
Radiation
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cheal, S. M., Ng, M., Barrios, B., Miao, Z., Kalani, A. K., & Meares, C. F. (2009). Mapping protein-protein interactions by localized oxidation: Consequences of the reach of hydroxyl radical. Biochemistry, 48(21), 4577-4586. https://doi.org/10.1021/bi900273j

Mapping protein-protein interactions by localized oxidation : Consequences of the reach of hydroxyl radical. / Cheal, Sarah M.; Ng, Mindy; Barrios, Brianda; Miao, Zheng; Kalani, Amir K.; Meares, Claude F.

In: Biochemistry, Vol. 48, No. 21, 02.06.2009, p. 4577-4586.

Research output: Contribution to journalArticle

Cheal, SM, Ng, M, Barrios, B, Miao, Z, Kalani, AK & Meares, CF 2009, 'Mapping protein-protein interactions by localized oxidation: Consequences of the reach of hydroxyl radical', Biochemistry, vol. 48, no. 21, pp. 4577-4586. https://doi.org/10.1021/bi900273j
Cheal, Sarah M. ; Ng, Mindy ; Barrios, Brianda ; Miao, Zheng ; Kalani, Amir K. ; Meares, Claude F. / Mapping protein-protein interactions by localized oxidation : Consequences of the reach of hydroxyl radical. In: Biochemistry. 2009 ; Vol. 48, No. 21. pp. 4577-4586.
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