Originpro 2015

All proteins including hTyrC_tr and OCA1B-related mutant variants were a subjected to equilibrium unfolding/refolding. Proteins were diluted to a final concentration of 1 μM in 10 mM phosphate buffer, pH 7.4 with urea concentrations from 0 to 8 M then incubated for 24h at RT. For equilibrium refolding experiments, 50 μM stocks of protein (final concentration 1 μM) in 8 M urea were incubated at RT for 5 h and then diluted with 10 mM phosphate buffer, pH 7.4 to 0–8 M urea and incubated for 24h at RT. Samples were excited at 285 nm and emission spectra was recorded at 300–400 nm. Both unfolding and refolding was monitored using the emission 360/320 nm ratio, which was plotted vs the urea concentration. The experimental curves were fitted with a Boltzmann function using OriginPro 2015 (Origin Lab Corporation, MA). Each unfolding/refolding curves were averaged in triplicate and normalized to show a change of unfolding fraction u from 0 to 1. The dependence of the unfolding fraction u on the urea concentration was converted to a free energy plot using the following formula : ΔG =−RT ln(u/(1−u)). Finally, the values of Gibbs free energy changes at zero urea concentration (‘physiological’ conditions) were obtained by the linear approximation of experimental points using the Origin Pro 2015 software (Origin Lab Corporation, MA).

Statistical analysis of the data was performed using OriginPro^® 2015 (OriginLab, Northampton, MA) using one-way ANOVA, followed by a Tukey test. In cases where P > 0.05, we reported no statistically significant difference between the two data sets in question.