Method: Native exchange NMR

Conditions: pH 6.5; 35.0 Celsius; Probes: 46

Related publication:
 PMID 7578123

Experiment details: "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 At different HX times, ranging from several minutes to about a week, aliquots (~0.6 mL) of the HX reaction were withdrawn and HX was quenched by dropping the pH* to 3.5 with DCl."

Protection threshold: strong protection

Sequence: KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV
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Method: Pulse labeling HDX NMR

Conditions: pH 4.0; 10.0 Celsius; Probes: 27

Related publication:
 PMID 2236032

Experiment details: "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."

Protection threshold: strong protection in I1

Sequence: KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Pulse labeling HDX NMR

Conditions: pH 4.0; 10.0 Celsius; Probes: 27

Related publication:
 PMID 2236032

Experiment details: "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."

Protection threshold: moderate protection in I1

Sequence: KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV
 CLICK TO DOWNLOAD SEQUENCE IN FASTA