Sample conditions. Two samples were used to record the data. Hsp60 reference sample: 26 μM hsp60 (416 μM monomer concentration) U-[2H, 15N], Met-[13CH3]. Hsp60 with HEWL: 26 μM hsp60 (416 μM monomer concentration) U-[2H, 15N], Met-[13CH3] and 52 μM HEWL U-[2H, 15N], Val/Leu-[13CH3]proS, Ile-[13CH3]δ1 (two equivalents of HEWL per hsp60 particle). All the samples were prepared in D2O buffer containing 100 mM MES (pH 6.5 uncorrected), 100 mM NaCl, 150 mM KCl, and 25 mM MgCl2.

Composition of the ATP regeneration system. ATP regeneration system was composed of 3 U of pyruvate kinase from Bacillus stearothermophilus, 0.1 mM ribulose-5-phosphate, and 220 mM PEP (Sigma-Aldrich).

Interleaved NMR experiments. The experiment was started by the addition of 10 mM ATP to the reaction mix, and the NMR tube was placed in the spectrometer at a temperature of 65°C. After an equilibration time of 5 min, 2D SOFAST-methyl-TROSY NMR spectra were recorded in interleaved mode with 1H 1D spectra.

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