Quenched-flow HDX NMR None highly protected within 250 ms EDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC

Quenched-flow experiments were carried out at 20 °C using a rapid mixing quenched-flow machine. In the first mixing step, refolding of protonated, unfolded ONC (in 6 M GdnHCl) was initiated by a 9-fold dilution into 50 mM sodium acetate buffer, pH 5.5. After defined refolding times (60 - 5000 ms), the samples were diluted 10-fold into D2O buffer (200 mM glycine buffer, 0.667 M GdnHCl, pD 11.0) to initiate the H/D exchange. The exchange was terminated after 8.3 ms by lowering the pH by a 2-fold dilution with D2O, containing 10 M acetic acid and 0.667 M GdnHCl.

Quenched-flow HDX NMR None protected after 250 ms EDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC

Quenched-flow experiments were carried out at 20 °C using a rapid mixing quenched-flow machine. In the first mixing step, refolding of protonated, unfolded ONC (in 6 M GdnHCl) was initiated by a 9-fold dilution into 50 mM sodium acetate buffer, pH 5.5. After defined refolding times (60 - 5000 ms), the samples were diluted 10-fold into D2O buffer (200 mM glycine buffer, 0.667 M GdnHCl, pD 11.0) to initiate the H/D exchange. The exchange was terminated after 8.3 ms by lowering the pH by a 2-fold dilution with D2O, containing 10 M acetic acid and 0.667 M GdnHCl.

Native exchange NMR None amids that persist to exchange for 24 hours EDWLTFQKKHITNTRDVDCDNIMSTNLFHCKDKNTFIYSRPEPVKAICKGIIASKNVLTTSEFYLSDCNVTSRPCKYKLKKSTNKFCVTCENQAPVHFVGVGSC

To assess native state H/D exchange in ONC, a series of 2D 15N-HSQC spectra was recorded for 3 days on an Avance II 600 NMR spectrometer after dissolving ONC in D2O buffer.