Live Cell Chemical Profiling of Temporal Redox Dynamics in a Photoautotrophic Cyanobacterium

Abstract

Protein reduction-oxidation (redox) modification is an important mechanism that allows microorganisms to sense environmental changes and initiate cellular responses. We have developed a quantitative chemical probe approach for live cell labeling and imaging of proteins that are sensitive to redox modifications. We utilize this in vivo strategy to identify 176 proteins undergoing ∼5-10-fold dynamic redox change in response to nutrient limitation and subsequent replenishment in the photoautotrophic cyanobacterium Synechococcus sp. PCC 7002. We detect redox changes in as little as 30 s after nutrient perturbation and oscillations in reduction and oxidation for 60 min following the perturbation. Many of the proteins undergoing dynamic redox transformations participate in the major components for the production (photosystems and electron transport chains) or consumption (Calvin-Benson cycle and protein synthesis) of reductant and/or energy in photosynthetic organisms. Thus, our in vivo approach reveals new redox-susceptible proteins and validates those previously identified in vitro.

Dinitrogenase-Driven Photobiological Hydrogen Production Combats Oxidative Stress in Cyanothece sp. Strain ATCC 51142

“…Mal-RP and IAM-RP are cysteine thiol redox-reactive chemical probes, and when used together, as in this study, are referred to as IM-RPs ( Fig. 3) (11,12). Samples were incubated at 30°C in the dark for 60 min.…”

“…TEV-biotin-N 3 (36 M), tris(2carboxyethyl)phosphine (TCEP) (2.5 mM), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (250 M) solubilized in a 4:1 tert-butanol-DMSO mixture, and CuSO 4 (50 M) were added to each sample and the samples were incubated at 24°C for 90 min, thereby connecting probe-labeled samples to TEV-biotin. Samples were then processed for streptavidin affinity purification, trypsin cleavage, and TEV protease cleavage, as previously described (11), with the exception that only 260 g of protein was enriched on 50 l of streptavidin agarose resin slurry.…”

“…P i , inorganic phosphate. system for peptide separation (LC-MS), as previously described (11,14). Data were acquired for 100 min, beginning 65 min after sample injection (15 min into gradient).…”

“…The tandem MS data were searched using the MS-GFϩ function, as previously described (11). Differential modifications of ϩ567.313 (IAM-RP) and ϩ678.345 (Mal-RP) were specified for cysteine probe modifications, and a differential modification of ϩ15.995 was specified for methionine oxidation.…”

“…51142 cells held under an Ar atmosphere were transitioned from N-limited chemostat growth to a N-depleted state and incubated with or without CO 2 . The cell dynamics were interrogated via a chemical probe-based chemoproteomic technique (11)(12)(13) to quantify which specific protein redox dynamics in vivo were correlated with H 2 production. Along with identifying the pro-teins that were reduced and probe labeled throughout each time course, we also used tandem protein cleavage techniques to identify the cysteine(s) responsible for conferring redox reactivity.…”