Pulse labeling HDX NMR and MS None refolding time constant < 180 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLVTLNPKLKSTGAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALPMIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIDV

Samples were prepared using a QFM-5 rapid-mixing quenched-flow device. Standard pulse-labeling procedure was performed, in which deuterated unfolded lysozyme (5 mg/ml in 3 M Gdm2HCl and 500 mM DTT) was exposed to a high pH labeling pulse after various refolding periods (3.5-2000 ms) at 20 °C in 20 mM sodium acetate buffer, pH 5.6.

Pulse labeling HDX NMR and MS None 180 < refolding time constant < 200 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLVTLNPKLKSTGAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALPMIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIDV

Samples were prepared using a QFM-5 rapid-mixing quenched-flow device. Standard pulse-labeling procedure was performed, in which deuterated unfolded lysozyme (5 mg/ml in 3 M Gdm2HCl and 500 mM DTT) was exposed to a high pH labeling pulse after various refolding periods (3.5-2000 ms) at 20 °C in 20 mM sodium acetate buffer, pH 5.6.

Pulse labeling HDX NMR and MS None refolding time constant > 200 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLVTLNPKLKSTGAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALPMIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIDV

Samples were prepared using a QFM-5 rapid-mixing quenched-flow device. Standard pulse-labeling procedure was performed, in which deuterated unfolded lysozyme (5 mg/ml in 3 M Gdm2HCl and 500 mM DTT) was exposed to a high pH labeling pulse after various refolding periods (3.5-2000 ms) at 20 °C in 20 mM sodium acetate buffer, pH 5.6.

Native exchange NMR None log(P) > 5 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLVTLNPKLKSTGAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALPMIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIDV

H/D exchange rates were measured using 2 mM 15N-labeled enzyme, on the basis of published resonance assignments. Exchange was initiated by a 10-fold dilution of a 2 mM 15N-labeled protein sample in 10 mM HEPES, pH 7.0, into a D2O solution at pH 5.6 or 4.4. Samples were subsequently concentrated 10-fold by ultrafiltration at 4 °C to give a final protein concentration of 2 mM; this procedure was repeated five times. Following exchange and concentration, the protein sample was subsequently analyzed by 2D NMR.

Native exchange NMR None 3 < log(P) < 5 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLVTLNPKLKSTGAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALPMIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIDV

H/D exchange rates were measured using 2 mM 15N-labeled enzyme, on the basis of published resonance assignments. Exchange was initiated by a 10-fold dilution of a 2 mM 15N-labeled protein sample in 10 mM HEPES, pH 7.0, into a D2O solution at pH 5.6 or 4.4. Samples were subsequently concentrated 10-fold by ultrafiltration at 4 °C to give a final protein concentration of 2 mM; this procedure was repeated five times. Following exchange and concentration, the protein sample was subsequently analyzed by 2D NMR.

Native exchange NMR None log(P) < 3 MVEINNQRKAFLDMLAWSEGTDNGRQKTRNHGYDVIVGGELFTDYSDHPRKLVTLNPKLKSTGAGRYQLLSRWWDAYRKQLGLKDFSPKSQDAVALQQIKERGALPMIDRGDIRQAIDRCSNIWASLPGAGYGQFEHKADSLIAKFKEAGGTVREIDV

H/D exchange rates were measured using 2 mM 15N-labeled enzyme, on the basis of published resonance assignments. Exchange was initiated by a 10-fold dilution of a 2 mM 15N-labeled protein sample in 10 mM HEPES, pH 7.0, into a D2O solution at pH 5.6 or 4.4. Samples were subsequently concentrated 10-fold by ultrafiltration at 4 °C to give a final protein concentration of 2 mM; this procedure was repeated five times. Following exchange and concentration, the protein sample was subsequently analyzed by 2D NMR.