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 language | English (US) |
|---|---|
| Pages (from-to) | 4577-4586 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 48 |
| Issue number | 21 |
| DOIs | |
| State | Published - Jun 2 2009 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
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 journal › Article
}
TY - JOUR
T1 - Mapping protein-protein interactions by localized oxidation
T2 - Consequences of the reach of hydroxyl radical
AU - Cheal, Sarah M.
AU - Ng, Mindy
AU - Barrios, Brianda
AU - Miao, Zheng
AU - Kalani, Amir K.
AU - Meares, Claude F.
PY - 2009/6/2
Y1 - 2009/6/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=66349091586&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=66349091586&partnerID=8YFLogxK
U2 - 10.1021/bi900273j
DO - 10.1021/bi900273j
M3 - Article
C2 - 19354299
AN - SCOPUS:66349091586
VL - 48
SP - 4577
EP - 4586
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 21
ER -