Quantification of Redox-Sensitive GFP Cysteine Redox State via Gel-Based Read-Out.

Abstract

To date, fluorescent protein biosensors are widely used in research. In vivo, they can be applied to dynamically monitor several physiological parameters in various subcellular compartments. Redox-sensitive green fluorescent protein2 (roGFP2) senses the glutathione redox potential via a disulfide bridge formed between neighboring beta-strands of its beta-barrel structure. As changes in redox state affect both excitation maxima of roGFP2 oppositely, sensor responses are ratiometric. The reaction mechanism of roGFP2 is well characterized and involves an intermediate S-glutathionylation step. Thus, roGFP2 is also used in enzymatic in vitro assays, e.g., assessing glutaredoxin kinetics. In addition to the fluorescent read-out, theroGFP2 redox state can also be determined by differential migration on a non-reducing SDS-PAGE. This read-out mode may be beneficial in some applications, e.g., if mass-spectrometric analysis of posttranslational cysteine modifications is desired. Here, we describe a protocol for gel-based fluorescent read-out of theroGFP2 redox state, as well as modification of free cysteines by maleimide-based reagents.