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.

Interactions of AsCy3 with Cysteine-Rich Peptides

“… 15 Since 2007, AsCy3 has been transformed into a super-resolution probe 16 and a membrane-permeable dye via substitution of the anionic sulfonate side chains for methyl esters, 17 and the monoarsenic variant has explored dithiol oxidation in bacteria. 18 Here we report that the initially described Cys 4 motif binds AsCy3 with only modest affinity and fluorescent enhancement, but that higher affinity (100-fold) and brightness (>3-fold) is seen with the expanded Cys 4 motif Cys-Cys-Lys-Ala-Glu-Ala-Ala-Lys-Ala-Glu-Ala-Ala-Lys-Cys-Cys. We hope that this information will aid researchers as they apply AsCy3 to characterize protein interactions on the cell surface and ultimately within the cytosol.…”

“…The observation that AsCy3 interacts comparably with peptides containing one or two Cys-Cys motifs suggests that only one Cys-Cys pair in the Cy3Tag sequence contributes to complex stability. 18 Indeed, the change in AsCy3 fluorescence emission (100 nM) in the presence of TagΔ2 (10 μM) is >60% of the enhancement observed with CyTag. This observation indicates that the second Cys-Cys pair contributes minimally, if at all, to AsCy3 fluorogenicity, and the dye may only be partially bound to all four cysteines ( Figure S1C in Supporting Information ).…”

Distance-Matched Tagging Sequence Optimizes Live-Cell Protein Labeling by a Biarsenical Fluorescent Reagent AsCy3_E

“…AsCy3_E has previously been shown to selectively label the Cy3TAG engineered near the C-terminus of the α-subunit of RNA polymerase expressed in living E. coli , permitting visualization of changes in cellular localization in response to metabolic conditions. 4 These prior experiments directly excited the AsCy3_E chromophore, which nonselectively excited both AsCy3_E bound to RNA polymerase as well as any dyes remaining within the cell. In the current experiments, we seek to quantitatively assess possible differences in the relative affinities of AsCy3_E binding to the Cy3TAG- or Cy3TAG+6-EGFP* constructs, as well as possible differences in their fluorescence lifetimes, which are directly related to the brightness of the Cy dyes.…”

“… 1 − 3 The small size and tetracoordinate linkage between the biarsenical probe and the protein backbone facilitate the reliable measurement of protein conformational states and protein–protein interactions. 4 − 13 …”

“… 18 Although originally synthesized as the sulfonate derivative AsCy3_S to increase water solubility, subsequent measurements demonstrated that the methoxyester-derivative AsCy3_E provides enhanced cellular permeability for live-cell imaging of the tagged proteins in prokaryotic and eukaryotic cells. 4 , 6 , 19 …”