Proteins were dialyzed in 20 mM phosphate buffer pH 7.4, 100 mM KF, 0.25 mM TCEP and diluted in this buffer to 0.2 mg.mL−1. CD spectra were recorded on a J-815 spectropolarimeter (Jasco (France); 1 mm path length quartz cell, ‘standard’ sensitivity, 0.5 nm bandwidth, 10 nm.min−1 scanning, 2 s digital integration time, 1 nm step resolution). The measuring chamber was maintained in nitrogen (17 L.min−1). Each spectrum was recorded as an average of 4 scans to reduce noise. After baseline correction, the mean residue ellipticity was calculated [θ]MRW,λ = MRW.θλ/10.d.c, where MRW is the mean residue weight (MRW = M/(N-1)), M is the molecular mass of the protein, N is the number of amino acids in the protein, θλ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm) of the cell, and c is the protein concentration (g.ml−1). The figure represents the CD spectra of wt (white circles) and Asp272Ala (black circles) hTEAD4217-434.