Cysteine sulfenic acid (Cys-SOH) is occurs transiently in some proteins in vivo that have redox-active cysteines as part of their normal catalytic function in oxidative damage control. Takanishi (2007) identify 32 E. coli proteins by LC-MS-MS that bind to a novel Cys-SOH trapping reagent, Yeast Yap1 transcription factor.
The hexahistidine-tagged Yap1 Cys-rich C-terminal domain formed mixed disulfide bonds preferentially with Cys-SOH residues after E. coli cells were treated with peroxide. Induced, Yap1-bound AhpC and OxyR were detected immunologically after nickel chromatography, DTT elution and SDS-PAGE gel electrophoresis, as expected.
32 bound proteins were sequenced by tandem mass spectrometry, including AhpC but not OxyR.
Four major bands were identified as AhpC, Bcp, Tpx, and DnaK. AhpC and Tpx were previously shown to form Cys-SOH intermediates at their active sites and AhpC, Tpx and Bcp all belong to the Pfam PF00578 AhpC-TSA (Thiol-Specific Antioxidant) family.
DnaK also bound to the Yap1 Cys598A control, so it is omitted from the gene set. Other proteins might be trapped non-specifically.
28 additional proteins were identified in seven regions of the gel after in situ digestion.
A fifth major band was a mixture of comigrating IscA, SufA, TrxA and YccK(TusE); all have redox-active cysteines.
It is likely that Yap1 is binding to highly reactive cysteines with low pKa sulfhydryl groups. PpiC was also present in this band, possibly due to a non-specific interaction.
Although presented in Table 1 (Takanishi, 2007) and indeed crosslinking may occur because the proteins have at least one Cys residue, 10 abundant proteins and proteins known to bind non-specifically were omitted from this Topic gene set (ClpP, DnaK, Hns, PpiC, RplK, RplF, RpoA, RpsA, Tsf, TufB).
In summary, of the 33 identified proteins (including OxyR), 23 are linked to this Topic,.
Six have known or probable Cys-SOH (AhpC, Tpx, Bcp, MsrA, MsrB, BtuE).
Ten have known or probable redox-active cysteines (AceF, GntY, HslO, IscA, IscU, NuoE, SufA, ThiI, TrxA, YccK).
Distinguishing the non-specific, Cys-SOH, and reactive-Cys Yap1-binding proteins among the seven unknowns (AceF, Adk, Def, Icd, NusA, YbiS, YgfZ) in this gene set requires additional biochemical work.
Although Def does bind proteins non-specifically, crystal structures show that Cys91 coordinates an Fe(2+) ion with His133 and His137, close to Cys130, so it is left in the gene set.
AceF, Adk, Icd and NusA may be contaminanting proteins.
YbiS is an uncharacterized periplasm protein of the ErfK family and is the only bound protein identified that is naturally in an oxidizing environment.
YgfZ is an interesting global regulator involved in a methylthio tRNA modification.
Since the method is sensitive enough to crosslink to the low abundance OxyR transcriptional regulator, similar rare Cys-reactive proteins could be specifically tested using a Western blot. Alternately, proteins to test, even foreign proteins, might be overexpressed before peroxide treatment to observe a new band appearing after target and probe co-induction.
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