HDX-folding competition by NMR None ln(P) > 1.0 RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRK__PFLKSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILDKALDLRILE

A 0.5 mL amount of a 2 mM sample of 15N-labeled CD2.D1 in H2O, containing 2.8 M GuHCl, was mixed against 8.3 vol of D2O, containing 25 mM sodium borate and 25 mM NaH2PO4, at the appropriate pH (not corrected), and 0.448 M Na2SO4, at 25C in a custom-built, rapid mixing device with a dead time of 2-3 ms (gives final concentrations of Na2SO4 and GuHCl of 0.4 and 0.3 M, respectively). Exchange was then quenched by the rapid addition (delay time = 2 s) of 6 mL of ice-cold D2O containing 100 mM sodium d3-acetate, pH 4.0.

HDX-folding competition by NMR None 0.5 < ln(P) < 1.0 RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRK__PFLKSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILDKALDLRILE

A 0.5 mL amount of a 2 mM sample of 15N-labeled CD2.D1 in H2O, containing 2.8 M GuHCl, was mixed against 8.3 vol of D2O, containing 25 mM sodium borate and 25 mM NaH2PO4, at the appropriate pH (not corrected), and 0.448 M Na2SO4, at 25C in a custom-built, rapid mixing device with a dead time of 2-3 ms (gives final concentrations of Na2SO4 and GuHCl of 0.4 and 0.3 M, respectively). Exchange was then quenched by the rapid addition (delay time = 2 s) of 6 mL of ice-cold D2O containing 100 mM sodium d3-acetate, pH 4.0.

HDX-folding competition by NMR None 0 < ln(P) < 0.5 RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRK__PFLKSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILDKALDLRILE

A 0.5 mL amount of a 2 mM sample of 15N-labeled CD2.D1 in H2O, containing 2.8 M GuHCl, was mixed against 8.3 vol of D2O, containing 25 mM sodium borate and 25 mM NaH2PO4, at the appropriate pH (not corrected), and 0.448 M Na2SO4, at 25C in a custom-built, rapid mixing device with a dead time of 2-3 ms (gives final concentrations of Na2SO4 and GuHCl of 0.4 and 0.3 M, respectively). Exchange was then quenched by the rapid addition (delay time = 2 s) of 6 mL of ice-cold D2O containing 100 mM sodium d3-acetate, pH 4.0.

Native exchange NMR None ΔG1 > 6.5 RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRK__PFLKSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILDKALDLRILE

Freeze-dried 15N-labeled CD2.D1 was dissolved to a concentration of 0.5 mM in D2O, containing 25 mM KH2PO4 (pH* 5.5), 0.2 M Na2SO4, 0.02% sodium azide and the appropriate concentration of Gu2HCl. For each concentration of Gu2HCl, a series of 1H-15N heteronuclear single quantum coherence (HSQC) spectra were acquired at appropriate time intervals in a 500 MHz spectrometer at 25 °C.

Native exchange NMR None 4 < ΔG1 < 6.5 RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRK__PFLKSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILDKALDLRILE

Freeze-dried 15N-labeled CD2.D1 was dissolved to a concentration of 0.5 mM in D2O, containing 25 mM KH2PO4 (pH* 5.5), 0.2 M Na2SO4, 0.02% sodium azide and the appropriate concentration of Gu2HCl. For each concentration of Gu2HCl, a series of 1H-15N heteronuclear single quantum coherence (HSQC) spectra were acquired at appropriate time intervals in a 500 MHz spectrometer at 25 °C.