Quenched-flow HDX NMR None refolding time constant < 30 ms LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

All experiments were carried out at 5+/-0.1 °C using a RQF-63 rapid mixing quenched-flow apparatus. Complete denaturation and exchange of the backbone amide protons with deuterium was achieved by dissolving CTX III (30 mg/ml) in 6 M deuterated guanidine hydrochloride in D2O at pH 6.6. Deuterated guanidine hydrochloride was obtained through repeated cycles of dissolving ultrapure GdnHCl in D2O followed by repeated lyophilization. Refolding of the denatured protein was initiated by 10-fold dilution with 50 mM glycine-d5 (pH 3.0) in H2O. At this pH, negligible labeling occurred. After variable refolding times ranging from 9.8 to 190.4 ms, the solution was diluted again to 10 times the initial protein volume with 0.2 M sodium borate (pH 9.6) to initiate labeling of the deuterated amides in CTX III with protons. After a lapse of 10 ms, the labeling pulse was stopped by a further 3.4-fold dilution of the initial protein volume with 1 M HCl in water. The final pH was about 3.2, at which the hydrogen/deuterium exchange (for the amides in native CTX III) was minimal.

Quenched-flow HDX NMR None 30ms < refolding time constant < 50 ms LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

All experiments were carried out at 5+/-0.1 °C using a RQF-63 rapid mixing quenched-flow apparatus. Complete denaturation and exchange of the backbone amide protons with deuterium was achieved by dissolving CTX III (30 mg/ml) in 6 M deuterated guanidine hydrochloride in D2O at pH 6.6. Deuterated guanidine hydrochloride was obtained through repeated cycles of dissolving ultrapure GdnHCl in D2O followed by repeated lyophilization. Refolding of the denatured protein was initiated by 10-fold dilution with 50 mM glycine-d5 (pH 3.0) in H2O. At this pH, negligible labeling occurred. After variable refolding times ranging from 9.8 to 190.4 ms, the solution was diluted again to 10 times the initial protein volume with 0.2 M sodium borate (pH 9.6) to initiate labeling of the deuterated amides in CTX III with protons. After a lapse of 10 ms, the labeling pulse was stopped by a further 3.4-fold dilution of the initial protein volume with 1 M HCl in water. The final pH was about 3.2, at which the hydrogen/deuterium exchange (for the amides in native CTX III) was minimal.

Quenched-flow HDX NMR None 50ms < refolding time constant < 100 ms LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

All experiments were carried out at 5+/-0.1 °C using a RQF-63 rapid mixing quenched-flow apparatus. Complete denaturation and exchange of the backbone amide protons with deuterium was achieved by dissolving CTX III (30 mg/ml) in 6 M deuterated guanidine hydrochloride in D2O at pH 6.6. Deuterated guanidine hydrochloride was obtained through repeated cycles of dissolving ultrapure GdnHCl in D2O followed by repeated lyophilization. Refolding of the denatured protein was initiated by 10-fold dilution with 50 mM glycine-d5 (pH 3.0) in H2O. At this pH, negligible labeling occurred. After variable refolding times ranging from 9.8 to 190.4 ms, the solution was diluted again to 10 times the initial protein volume with 0.2 M sodium borate (pH 9.6) to initiate labeling of the deuterated amides in CTX III with protons. After a lapse of 10 ms, the labeling pulse was stopped by a further 3.4-fold dilution of the initial protein volume with 1 M HCl in water. The final pH was about 3.2, at which the hydrogen/deuterium exchange (for the amides in native CTX III) was minimal.

Native exchange NMR None log(P) > 4.0 LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

H/D exchange measurements in CTX III and CBTX were monitored using the magnitude COSY spectra recorded at 25°C (pH 3.4) using a Bruker DMX-600 NMR spectrometer. The samples for exchange kinetics of the amide protons in the proteins (CBTX and CTX III) were prepared by dissolving the lyophilized proteins in deuterated buffer at pD 3.6. The concentrations of the proteins (CBTX and CTX III) were 2.0 mM.

Native exchange NMR None 2.0 < log(P) < 4.0 LKCNKLVPLFYKTCPAGKNLCYKMFMVATPKVPVKRGCIDVCPKSSLLVKYVCCNTDRCN

H/D exchange measurements in CTX III and CBTX were monitored using the magnitude COSY spectra recorded at 25 °C (pH 3.4) using a Bruker DMX-600 NMR spectrometer. The samples for exchange kinetics of the amide protons in the proteins (CBTX and CTX III) were prepared by dissolving the lyophilized proteins in deuterated buffer at pD 3.6. The concentrations of the proteins (CBTX and CTX III) were 2.0 mM.