Mutagenesis and Purification of Fluorescent Proteins

The K206A mutation was introduced by mutagenesis in SCFP3A, mTurquoise and mTurquoise2 on the pRSET vector (primers listed in Supplementary Table S6). After verification by sequencing, the coding sequence of the CFP variants was transferred into pQE60–Cerulean7 (link), using the NcoI and BsrGI restriction sites to replace Cerulean. His-tagged recombinant proteins meant for crystallization were expressed in E. coli BL21 CodonPlus (DE3) RIL cells (Stratagene) in autoinduction medium, at 27 °C for 24 h on the RoBioMol platform of the Institut de Biologie Structurale. Cells were lysed by sonication in the presence of 20 mM Tris (pH 8.0) and 500 mM NaCl with EDTA-free protease inhibitors (Complete, Roche). His-tagged proteins were purified on a Ni-NTA Superflow column (Qiagen) and eluted with 100 mM imidazole in the buffer described above. Fractions containing purified proteins were pooled, dialysed against 20 mM Tris (pH 8.0), and concentrated to 38–85 mg ml⁻¹. Site-directed mutagenesis of pRSET-mTurquoise on position 146, 165 or 220 were performed using the primers listed in Supplementary Table S6. For spectroscopic characterization, proteins were expressed using the pRSET vectors expressed in BL21 (DE3) cells and purified as described10 (link).