Figure 2

Characterisation of N- and C-terminal domains. (a) Tryptophan fluorescence of NTD2. The ratio between fluorescence at 338 and 352 nm as a function of pH is shown. The full fluorescence spectra at different pH values is included as an inset, with pH indicated by colour (red at pH 8.2 through to green at pH 5.5). Error bars show the standard deviation of three technical replicates. (b) Samples were run at pH 8 (red) and pH 5.5 (green), either in the presence of 300 mM NaCl (solid lines) or in its absence (dashed lines). With NaCl, peak elution corresponded to monomer and dimer species at pH 8 and pH 5.5, respectively. In the absence of NaCl, peaks were shifted to higher apparent molecular weights, possibly indicating the formation of larger multimers. Following elution, samples in the absence of salt appeared cloudy, suggesting aggregation. mAU stands for milli-absorbance units. (c) Two independent reducing and non-reducing SDS-PAGE gels showing the purification of CTD1 by nickel immobilized metal affinity chromatography. S: Soluble, I: Insoluble, FT: Flow-through, P: Purified. A band corresponding to a CTD1 monomer is observed under reducing conditions, while a band corresponding to a CTD1 dimer is observed under non-reducing conditions (red arrows). (d) Thermal stability of CTD1 with or without NaCl or KPi, as determined by dynamic scanning fluorometry (Supplementary Fig. S2). As pH decreases, there is a decrease in CTD1 thermal stability. Error bars show the standard deviation of three technical replicates.