We mixed fibril samples with increasing volumes of urea stock solutions (see below) and decreasing volumes of buffer to create denaturation series of linearly increasing urea concentration and constant protein concentration of 40µM for I22A, I29A, I53A, I77A, I82A and one WT reference and 30 µM for all other variants as well as four WT references. In order to maintain a solution pH of 2 constant throughout the whole denaturation series, we prepared an 8 M urea stock solution by dissolving 12 g of urea (SigmaAldrich) in 16 ml of concentrated buffer with added HCl, to yield a final concentration of 10 mM glycine hydrochloride and 16 mM HCl. The HCl is necessary, as the urea is weakly basic. We confirmed the urea concentration by refractive index measurements with an Ataga R-5000 refractometer. In order to keep the ionic strength constant in all samples, we added NaCl to the 10 mM glycine HCl buffer to a final concentration of 16 mM, in order to maintain overall ionic strength . We allowed samples to equilibrate for two weeks at 25 • C, which has been demonstrated to be sufficient for these conditions . We distributed the PI3K-SH3 variants into four experimental sets, each containing an internal WT reference as a control of reproducibility. The I22A, I29A, I53, I77A and I82A mutants are experimental set 1. We measured the I77A, I82A mutants and the WT reference in a 26 sample series from 0 to 6.4 M urea, while I22A, I29A and I53 mutants are measured in a 24 sample series from 0 to 6 M urea. L11V, V32A and L40V variants are experimental set 2, the T62S, T63S and V74A variants are experimental set 3 and the A3G, Y6A, Y8A and L11A variants are experimental set 4. We measured the variants and WT samples of set 2, 3 and 4 in a 16 sample series from 0 to 6.4 M urea.