UV-CD Measurements:

Samples were prepared for UV-CD measurements by carrying out a potentiometric titration as previously described ²⁹, except that at regular increments throughout the titration, 40 μL aliquots were removed from the sample being titrated and set aside for UV-CD measurements. Subsequently, these aliquots were loaded sequentially into a 0.1 mm pathlength demountable quartz cuvette, and UV-CD spectra were collected at 20°C using a JASCO J-810 spectropolarimeter. All UV-CD spectra were the average of six scans from 260–190 nm, with a 1 nm step, a 2 second response time, and 50 nm/min scanning speed. The UV-CD spectra measured for background solution at low and high pH were identical within error, so the low pH background was subtracted from all spectra. The resulting ellipticity was used to calculate the mean residue ellipticity ([θ], units of deg cm²/dmol residue) using Equation (3):

where θ=ellipticity (mdeg, machine units), N is the number of residues, N–1 is the number of backbone amides, Lmm is the path length of the cuvette (mm), and CM is the protein concentration in molar units.