FIGURE 3.

Thermofluor assays of the purified PilF wild-type complex. Thermofluor assays were performed at pH 8.5 (50 mm Bicine buffer with 200 mm NaCl). 20 μl of purified PilF complex (0.1 μg/μl) were used in thermofluor assays. The melting curve of PilF wild-type complex was measured in Rotor Gene Q5 Plex HRM thermo cycler (A). SYPRO® Orange was used to measure fluorescence in the HRM channel with the excitation wavelength at 460 nm and the emission wavelength at 510 nm. The first derivative of the melting curve is shown in B. Two transition temperatures were observed at 80.8 ± 0.8 °C and 90.5 ± 0.0 °C. The effect of cysteine mutations on AMP-PNP- and ADP-mediated stabilization of PilF complexes is shown in C. Unfolding of the PilF complexes was measured by thermofluor assays. The bars indicate the first melting transitions of different PilF complexes in the absence or in the presence of 5 mm AMP-PNP and 2.5 mm ADP, respectively. Black, wild type; white, C1A; light gray, C2A; middle gray, HC2A; dark gray, C3A; vertical stripes, C4A; diagonal stripes, 2CysA; horizontal stripes, 3CysA; black and white boxes, 4CysA.