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3 Material and Methods Protein Preparation DsLOV and the two DsLOV variants were heterologeously produced in E. coli BL21 (DE3), cultivated in auto-induction medium (TB, 0.2 % lactose, 0.05 % glucose, 50 μg/ml kanamycin) [9] and purified as described previously [14]. In brief, cells were harvested and approx. 5 g (wet weight) were dissolved in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and subsequently lysed by passing the cell suspension four times through a french pressure cell (Thermo Scientific, Waltham, MA). The soluble cell free extract was obtained by centrifugation (9200 × g, 30 min). The His6-tagged DsLOV proteins were subsequently purified by metal affinity chromatography using a superflow Ni-NTA resin (QIAGEN, Hilden, Germany). Elution was performed using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Protein containing fractions were pooled, and the elution buffer was exchanged to 10 mM sodium phosphate, pH 8.0, supplemented with 300 mM NaCl. Desalting and concentration was achieved using Vivaspin centrifugal concentrator units (Vivascience AG, Sartorius, Goettingen, Germany) with a cut-off of 10 kDa. Thermal unfolding experiments The DsLOV protein samples were subjected to a linear thermal ramp (1 °C/min, from 20 °C to 95 °C), and fluorescence at 350 nm and 330 nm was collected at a rate of 10 data points per minute. Unfolding transition midpoints were determined automatically from the first derivative of the fluorescence ratio (F350/F330). References 1. Lakowicz, J.R., Principles of fluorescence spectroscopy. 3rd ed. 2006, New York: Springer. xxvi, 954 p. 16-19 2. Krauss, U., et al., Distribution and phylogeny of light-oxygen-voltage-blue-light-signaling proteins in the three kingdoms of life. J Bacteriol, 2009. 191(23): p. 7234-42. 3. Salomon, M., et al., Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry, 2000. 39(31): p. 9401-10. 4. Jentzsch, K., et al., Mutual exchange of kinetic properties by extended mutagenesis in two short LOV domain proteins from Pseudomonas putida. Biochemistry, 2009. 48(43): p. 10321-33. 5. Endres, S., et al., Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae. BMC Microbiology, 2015. 15.