Assignment of HEWL Ile-δ1, Val and Leu-proS methyl groups. Folded HEWL assignment was obtained from the published assignment (40). Unfolded HEWL assignment was transferred from the folded HEWL using a methyl-TROSY-EXSY experiment recorded at 65°C, where both unfolded and folded populations of HEWL can be observed (fig. S5D).

Assignment of hsp60 Met-ε and Val-γ2 methyl groups. Each mutant sample (40 μl) was loaded in a 1.7-mm tube, and the NMR experiments were recorded on a Bruker Avance III HD spectrometer operating at a 1H frequency of 850 MHz and equipped with a 1.7-mm TCI MicroCryoProbe. The 2D SOFAST-methyl-TROSY NMR experiments (18) were recorded at 75°C with an adjusted duration depending on the final concentration of each mutant (experimental time ranging from 60 to 120 min per sample). For each mutant, spectra (fig. S1) were recorded for the apo state, App-NHp–bound state (1 mM), or ADP-bound state (1 mM). Analysis and comparison of the complete library of mutant spectra allowed the assignment of 33 valines (70%) and 15 methionines (100%). To obtain the missing assignment and confirm the assignment obtained by mutagenesis, a 3D HMQC-NOESY-HMQC experiment was recorded with a 47 μM (0.75 mM monomer) U-[2H, 15N], Met-[13CH3], Val-[13CH3]proS sample of hsp60 (fig. S2). Comparison of the NOE cross peaks with the model of the structure of hsp60 allowed to confirm the mutagenesis assignment and to assign 43 valines (92%) and 15 methionines (100%).

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