Cysteine thiols are susceptible to a range of oxidative modifications, which make it a powerful chemical agent for signaling and regulatory processes in microbes. Cysteine thiol reduction and oxidation (redox) is a common mechanism used by microbes to sense environmental perturbations and initiate responses. Photosynthetic cyanobacteria are of great interest due to their potential to generate biofuels, but in order to fully realize their capacity we believe that the regulatory redox dynamics of protein cysteine thiols must first be understood. We hypothesized that there is widespread regulation of proteins involved in critical metabolic pathways, including those important to the synthesis of high-value small molecules, biofuel precursors, and/or hydrogen. A critical caveat to investigating thiol redox status is the limitation imposed by cell lysis, in which near immediate oxidation upon lysis destroys the native redox status. Therefore, we developed novel chemical probe strategies for targeting reduced protein cysteine thiols in living cells, and used an existing chemical probe to target sulfenic acid formation. We characterized redox dynamics in multiple nutrient conditions including C and N-limitation, high light and O2 stress, light to dark transitions, and H2 production. By parsing proteins that undergo redox reactions and those highly sensitive to ROS stress from the rest of the proteome, we can achieve a fuller understanding of photoautotrophic regulatory dynamics. Along with identifying key protein targets involved in photosynthesis, biosynthesis, electron transport and transcription regulation, we also identified a majority of the sites of probe labeling by applying sequential peptide cleavage techniques. Additionally we carried out complementary global proteomic, transcriptomic, confocal laser microscopy, and photophysiology measurements.  In total, our comprehensive approach provides a more thorough understanding of redox signaling that will facilitate optimization of cellular output.

Redox based protein profiling in live cyanobacteria cells to characterize dynamics associated with biofuel production

248th National Meeting (2014)

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