Simultaneous monitoring of CD, trp fluorescence, and SLS during isothermal urea denaturation at 25°C of hNBD1-Δ(RI,RE) ± F508del. Protein samples (2 μM) in the absence or presence of 8 M urea were progressively mixed every 800 seconds to achieve the indicated concentration of urea, using an autotitrator for CD measurements or an equivalent kinetic protocol for fluorescence and SLS measurements. The buffer contained 150 mM NaCl, 0.5 mM MgCl2 10% glycerol, 10% ethylene glycol, 0.8 mM TCEP, 20 mM Na-HEPES, pH 7.5. Data for F508 and F508del constructs are shown in black and gray, respectively. The buffer used to purify and store the protein, which is catalytically inactive,– introduces 30 μM Mg-ATP. Additional Mg-ATP was added to the indicated final concentration. CD (top) was monitored at 230 nm instead of 222 nm to enable use of a higher concentration of Mg-ATP without interference from nucleotide absorbance. Intrinsic trp fluorescence (middle) was excited at 297 nm and monitored at 340 nm. SLS at 297 nm (bottom) was measured at a 90° angle simultaneously with trp fluorescence in a T-format fluorimeter (see Methods). SLS counts are background-subtracted for scattering from the protein-free 0 M urea buffer for measurements in the fluorimeter, so the ratio of the SLS signal at a given urea concentration to that at the start of the titration gives a rough estimate of weight-averaged aggregation state. Note, however, that this calculation underestimates the aggregation state at higher urea concentrations because it ignores the effect of urea in reducing protein contrast.