title: Synthesis and application of an environmentally insensitive Cy3-based arsenical fluorescent probe to identify adaptive microbial responses involving proximal dithiol oxidation.
abstract: Reversible 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.
authors: Biological Sciences Division, Fundamental Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.
authors: Na
authors: N
authors: Fu
authors: Dian
authors: D
authors: Su
authors: John R
authors: JR
authors: Cort
authors: Baowei
authors: B
authors: Chen
authors: Yijia
authors: Y
authors: Xiong
authors: Wei-Jun
authors: WJ
authors: Qian
authors: Allan E
authors: AE
authors: Konopka
authors: Diana J
authors: DJ
authors: Bigelow
authors: Thomas C
authors: TC
authors: Squier
mesh_terms: D001151:Arsenic / Q000737:chemistry / Q000378:metabolism*; D002232:Carbocyanines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D046940:Synechococcus / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; C089187:cyanine dye 3; D001151:Arsenic