HDX-folding competition by NMR None KUI(loc) > 4 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLNGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

NMR-detected H/D exchange/ folding competition from the urea unfolded state with ultrarapid microfluidic mixer. The protein was initially unfolded in D2O (pD 2.0, 3 M urea) and rapidly mixed with a 4-fold excess of H2O refolding buffer at alkaline pH.

HDX-folding competition by NMR None 3 < KUI(loc) < 4 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLNGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

HDX-folding competition by NMR None 2 < KUI(loc) < 3 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLNGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

Native exchange NMR None P > E08 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

Oxidized horse cyt c at 20 °C, in D2O and 50 mM sodium phosphate, pD 7.0.

Native exchange NMR None E06 < P < E08 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

Oxidized horse cyt c at 20 °C, in D2O and 50 mM sodium phosphate, pD 7.0.

Native exchange in partially folded state by NMR None P > 500 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

A 6 mM solution of oxidized horse heart c was prepared by dissolving the lyophilized protein in 1.5 M NaCl solution in H2O adjusted to pH 2.2 by addition of HCI. Hydrogen-deuterium exchange was initiated by transferring the protein into an unbuffered D2O solution of 1.5 M NaCl adjusted to pD 2.2 (uncorrected electrode reading), spinning Sephadex G-25 columns being used (3 mL bed volume, 30 s centrifugation at 4OOg). The solution was incubated at 20 °C, and 0.5 mL aliquots were collected after H-D exchange times ranging from 2 min to 500 h. The reaction was quenched in a at 4 °C with a D2O solution of 50 mM sodium phosphate and 50 mM ascorbate at pD 5.3.

Native exchange in partially folded state by NMR None 100 < P < 500 GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

Pulse labeling HDX NMR None high protection within 100 ms GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

All experiments were performed at 10 °C. Oxidized cytochrome c (type VI from horse heart, purchased from Sigma) was initially dissolved in D2O at pD 6.0 containing 4.2 M guanidine hydrochloride (GuHCl) as a denaturant. These conditions were shown to produce complete unfolding of cytochrome c, as judged by circular dichroism and hydrogen exchange, with the exception of a limited amount of residual structure in the vicinity of the His 18 haem ligand. Initial protein concentration was 6 mM. Refolding was initiated in the first mixer (A) by rapid 6-fold dilution with 0.1 M acetate at pH 6.2 in H2O. The resulting GuHCl concentration (0.7 M) is well below the unfolding transition. After variable refolding times (tf), the solution was combined in a second mixer (B) with an equal volume of an H2O buffer (0.1 M sodium phosphate, 0.1 M glycine) at pH 9.3 (final pH determined in a separate control). Under these pulse conditions, freely accessible amide protons exchange in about 1 ms. The labelling pulse was terminated after 50±10 ms by a rapid pH change to 5.3, accomplished by injecting the solution at the exit of the mixing apparatus into a reservoir of quench buffer (0.3 M sodium citrate, 0.05 M sodium ascorbate, 0 °C) under vigorous stirring (C). The quench conditions favour slow H-exchange (intrinsic exchange times are ~10 s) and rapid refolding. The ascorbate in the quench solution serves both as pH buffer and reducing agent for cytochrome c, taking advantage of the fact that many amide protons exchange much more slowly in the reduced form of the protein. This procedure, with H2O present in the refolding period, was used for experiments at refolding times tf less than 1 s.

Pulse labeling HDX NMR None extensive protection (>40%) during the 5 ms dead-time GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE

Pulsed NH exchange experiments were performed on a QFM-5 quenched flow module at 10 °C, following published procedures. Oxidized cyt c (6 mM) was unfolded in an unbuffered D2O solution containing 4.2 M deuterated GuHCl adjusted to pD 5. Under these conditions, all backbone protons exchange rapidly with deuterons from the solvent. Refolding was initiated by 6-fold dilution of the denatured protein with refolding buffer (100 mM in adjusted to yield final pH of 5. The resulting refolding conditions (0.7 M GuHCl, pH 5, 10 °C, 83% H2O/17% D2O) are well below the equilibrium unfolding transition for cyt c. For the refolding periods used (5 ms to 60 s), D-H exchange is negligible even for unprotected amide groups. The D-H exchange reaction was initiated by rapidly mixing the partially refolded protein solution with an equal volume of 100 mM glycine buffer in H2O at pH 9.8, resulting in a jump to pH 9.5. The 32-ms labeling pulse was terminated by lowering the pH to 5.3 in a final 1:l mixing step with quench buffer (300 mM citrate at pH 5.3, containing 50 mM ascorbate as a reducing agent).