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Synthesis and Application of an Environmentally Insensitive Cy3-Based Arsenical Fluorescent Probe To Identify Adaptive Microbial Responses Involving Proximal Dithiol Oxidation
journal contribution
posted on 2016-02-19, 19:39 authored by Na Fu, Dian Su, John R. Cort, Baowei Chen, Yijia Xiong, Wei-Jun Qian, Allan E. Konopka, Diana
J. Bigelow, Thomas C. SquierReversible disulfide
oxidation between proximal cysteines in proteins
represents a common regulatory control mechanism to modulate flux
through metabolic pathways in response to changing environmental conditions.
To enable in vivo measurements of cellular redox
changes linked to disulfide bond formation, we have synthesized a
cell-permeable thiol-reactive affinity probe (TRAP) consisting of
a monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3)
to trap and visualize dithiols in cytosolic proteins. Alkylation of
reactive thiols prior to displacement of the bound TRAP_Cy3 by ethanedithiol
permits facile protein capture and mass spectrometric identification
of proximal reduced dithiols to the exclusion of individual cysteines.
Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen
and light levels in the photosynthetic microbe Synechococcus sp. PCC7002, we observe large decreases in the abundance of reduced
dithiols in cellular proteins, which suggest redox-dependent mechanisms
involving the oxidation of proximal disulfides. Under these same growth
conditions that result in the oxidation of proximal thiols, there
is a reduction in the abundance of post-translational oxidative protein
modifications involving methionine sulfoxide and nitrotyrosine. These
results suggest that the redox status of proximal cysteines responds
to environmental conditions, acting to regulate metabolic flux and
minimize the formation of reactive oxygen species to decrease oxidative
protein damage.
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redox statusdithiolvivo measurementsProximal Dithiol OxidationReversible disulfide oxidationdecrease oxidative protein damagemonosubstituted cyanine dye derivatizedPCCmethionine sulfoxidedisulfide bond formationIdentify Adaptive Microbial Responsesreactive oxygen speciesreactive thiolscytosolic proteinslight levelscysteineTRAPgrowth conditionsredox changescontrol mechanismphotosynthetic microbe Synechococcus sp
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