| Products/Services Used | Details | Operation |
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| Gene Synthesis | Point mutations were introduced by PCR with?Pfu?polymerase (Promega) using the mutagenesis primers listed in Supplementary Table?10?and the double stop-codon construct of pQE30/SCGRX7?(ref.?11) as template. Following the digestion of the methylated template DNA by?DpnI (NEB), plasmids were transformed into chemically competent?E. coli?XL1-Blue cells. Correct mutations and sequences were confirmed for all constructs by sequencing both strands (SEQ-IT). Codon- and mRNA structure-optimized genes?SCGRX7WP,?SCGRX7loop,?SCGRX7WP+loop,?HSGRX5C122S,?HSGRX5RR,?HSGRX5loop, and?HSGRX5RR+loop?were synthesized (Genscript) and either subcloned into the?EcoRI and?XhoI restriction sites of p416TEF/roGFP2?(ref.?45) for roGFP2 measurements in yeast or PCR-amplified using the primers in Supplementary Table?10?and subcloned into the?BamHI and?HindIII restriction sites of pQE30 for heterologous expression in?E. coli. Please note that all?HSGRX5?constructs encode C122S mutants that lack the mitochondrial presequence and start with residue Ala32. | Get A Quote |
Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed... More
