Fluorescent Labeling of ctPrp43

To generate two maleimide-reactive fluorescence-labeling sites in ctPrp43, eight native cysteine residues had to be considered. C148, C214, and C377 are buried inside the protein and inaccessible to the coupling group. C303, C323, C441, C508 and C543 are surface exposed and therefore accessible to the fluorescence dye. They were replaced by site directed mutagenesis (C303T, C323V, C441A, C508A and C543S). The labeling sites were generated by introducing cysteine residues at position K170 in the RecA1 and E602 in the WH. The mutant protein was expressed and purified as described in (18 (link)). The purified protein was mixed with Cy3-maleimeide and Cy5-maleimide (Cytiva), dissolved in dimethylsulfoxide at a molar ratio of 1:2:2 (protein:Cy3:Cy5) and incubated for 30 min at 20°C. Excess dye was removed by Ni-sepharose affinity chromatography, labeled protein was eluted in 50 mM Tris/HCl (pH 7.5), 400 mM NaCl, 5% (v/v) glycerol, 2 mM MgCl₂, 250 mM imidazole. The labeled protein was dialyzed twice against 50 mM Tris–HCl, pH 7.5, 300 mM KCl, 3 mM MgCl₂ using Slide-A Lyzer Dialysis Cassette G2 3.5K (Thermo) for 1 h at 4°C. The labeled protein was concentrated to final concentrations between 40 and 70 μM (Amicon Ultra 50K, Millipore).