Native exchange NMR None strong protection KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

Prior to the HX reaction, RNase A was lyophilized from aqueous solution at pH 6.5 and sucrose was lyophilized twice from D2O solutions at pH* 6.5. HX reaction was initiated by dissolving the lyophilized protein into D2O. At different HX times, ranging from several minutes to about a week, aliquots (~0.6 mL) of the HX reaction were withdrawn and HX was quenched by dropping the pH* to 3.5 with DCl.

Pulse labeling HDX NMR None strong protection in I1 KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

The deuterated RNase A was unfolded in an unfolding buffer (2.65 M guanidine hydrochloride/40 mM glycine, in D2O at a final pH of 2). Refolding of the unfolded RNase A solution (60-70 mg of RNase A per ml) at pH 4 was initiated by diluting 10.5-fold into a refolding buffer (0.442 M sodium sulfate/0.055 M sodium formate, in H2O at pH 4.25). At different times after beginning refolding, the exchange pulse was initiated by diluting 1.5-fold into an exchange buffer [0.4 M sodium sulfate/0.25 M guanidine hydrochloride/0.1 M (final) glycine, in H2O] so that the final pH was 9 or 10. The 37-msec exchange pulse was terminated by diluting 1.33-fold into a quench buffer (0.4 M sodium sulfate/0.25 M guanidine hydrochloride/0.1 M sodium formate), so that the final pH was 2.9. The refolding reaction was then allowed to go to completion (10 min) at this pH. The mixing dead time was 5 msec for each of the three mixing events described above. For the zero time point, the exchange pulse was applied directly to the unfolded protein solution.

Pulse labeling HDX NMR None moderate protection in I1 KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

The deuterated RNase A was unfolded in an unfolding buffer (2.65 M guanidine hydrochloride/40 mM glycine, in D2O at a final pH of 2). Refolding of the unfolded RNase A solution (60-70 mg of RNase A per ml) at pH 4 was initiated by diluting 10.5-fold into a refolding buffer (0.442 M sodium sulfate/0.055 M sodium formate, in H2O at pH 4.25). At different times after beginning refolding, the exchange pulse was initiated by diluting 1.5-fold into an exchange buffer [0.4 M sodium sulfate/0.25 M guanidine hydrochloride/0.1 M (final) glycine, in H2O] so that the final pH was 9 or 10. The 37-msec exchange pulse was terminated by diluting 1.33-fold into a quench buffer (0.4 M sodium sulfate/0.25 M guanidine hydrochloride/0.1 M sodium formate), so that the final pH was 2.9. The refolding reaction was then allowed to go to completion (10 min) at this pH. The mixing dead time was 5 msec for each of the three mixing events described above. For the zero time point, the exchange pulse was applied directly to the unfolded protein solution.