Method: Quenched-flow HDX NMR

Conditions: pH 7.8; 20.0 Celsius; Probes: 31

Related publication:
 PMID 11847289

Experiment details: "Denatured perdeutero hisactophilin (8.0 mg/mL in 6.4 M urea-D4, 250 mM potassium phosphate buffer at pH 7.8, 5 mM DTT, 5 mM EDTA, D2O) was diluted fivefold with D2O, incubated for a series of set refolding times, pH-pulsed using 5 volumes of 62.5 mM glycine/sodium glycinate in H2O (final pH was 9.52 for 29 ms), and pH-quenched with 4 volumes of 112.5 mM triethanolamine buffer in H2O (final pH was 7.7). The sample was immediately concentrated and exchanged by ultrafiltration into 50 mM triethanolamine buffer at pH 7.8 in D2O. Final sample volumes (0.5 mL) contained 5mg/mL hisactophilin. NMR experiments were performed within 2 h after concentration."

Protection threshold: Fast protection rate s(-1) > 20

Sequence: MGNRAFKSHHGHFLSAEGEAVKTHHGHHDHHTHFHVENHGGKVALKTHCGKYLSIGDHKQVYLSHHLHGDHSLFHLEHHGGKVSIKGHHHHYISADHHGHVSTKEHHDHDTTFEEIII
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Quenched-flow HDX NMR

Conditions: pH 7.8; 20.0 Celsius; Probes: 31

Related publication:
 PMID 11847289

Experiment details: "Denatured perdeutero hisactophilin (8.0 mg/mL in 6.4 M urea-D4, 250 mM potassium phosphate buffer at pH 7.8, 5 mM DTT, 5 mM EDTA, D2O) was diluted fivefold with D2O, incubated for a series of set refolding times, pH-pulsed using 5 volumes of 62.5 mM glycine/sodium glycinate in H2O (final pH was 9.52 for 29 ms), and pH-quenched with 4 volumes of 112.5 mM triethanolamine buffer in H2O (final pH was 7.7). The sample was immediately concentrated and exchanged by ultrafiltration into 50 mM triethanolamine buffer at pH 7.8 in D2O. Final sample volumes (0.5 mL) contained 5mg/mL hisactophilin. NMR experiments were performed within 2 h after concentration."

Protection threshold: 10 < fast protection rate s(-1) < 20

Sequence: MGNRAFKSHHGHFLSAEGEAVKTHHGHHDHHTHFHVENHGGKVALKTHCGKYLSIGDHKQVYLSHHLHGDHSLFHLEHHGGKVSIKGHHHHYISADHHGHVSTKEHHDHDTTFEEIII
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Quenched-flow HDX NMR

Conditions: pH 7.8; 20.0 Celsius; Probes: 31

Related publication:
 PMID 11847289

Experiment details: "Denatured perdeutero hisactophilin (8.0 mg/mL in 6.4 M urea-D4, 250 mM potassium phosphate buffer at pH 7.8, 5 mM DTT, 5 mM EDTA, D2O) was diluted fivefold with D2O, incubated for a series of set refolding times, pH-pulsed using 5 volumes of 62.5 mM glycine/sodium glycinate in H2O (final pH was 9.52 for 29 ms), and pH-quenched with 4 volumes of 112.5 mM triethanolamine buffer in H2O (final pH was 7.7). The sample was immediately concentrated and exchanged by ultrafiltration into 50 mM triethanolamine buffer at pH 7.8 in D2O. Final sample volumes (0.5 mL) contained 5mg/mL hisactophilin. NMR experiments were performed within 2 h after concentration."

Protection threshold: 4 < fast protection rate s(-1) < 10

Sequence: MGNRAFKSHHGHFLSAEGEAVKTHHGHHDHHTHFHVENHGGKVALKTHCGKYLSIGDHKQVYLSHHLHGDHSLFHLEHHGGKVSIKGHHHHYISADHHGHVSTKEHHDHDTTFEEIII
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Native exchange NMR

Conditions: pH 5.9-9.7; 17.4 Celsius; Probes: 37

Related publication:
 PMID 11802717

Experiment details: "For measurement of the pH dependence of amide exchange rates, purified protein was exchanged into 50 mM potassium phosphate buffer at pH 5.9, 6.8, and 7.8 or 50 mM glycine buffer at pH 8.7 and 9.7 and then lyophilized. H/D exchange was initiated by redissolving the sample in D2O to the same volume that existed prior to lyophilization. The slow exchange core is defined by the authors as the collection of residues with the slowest average exchange rate, the strongest dependence of exchange on denaturant concentration, and the strongest temperature dependence for exchange rates."

Protection threshold: Slow exchange core

Sequence: MGNRAFKSHHGHFLSAEGEAVKTHHGHHDHHTHFHVENHGGKVALKTHCGKYLSIGDHKQVYLSHHLHGDHSLFHLEHHGGKVSIKGHHHHYISADHHGHVSTKEHHDHDTTFEEIII
 CLICK TO DOWNLOAD SEQUENCE IN FASTA