Method: Pulse labeling HDX NMR

Conditions: pH 5.8; 25.0 Celsius; Probes: 51

Related publication:
 PMID 18779573

Experiment details: "A solution containing the fully unfolded apo-myoglobin (80 M) at pH 2.2 in HCl/H2O was placed in syringe P. Refolding and H/D exchange was performed at room temperature. The refolding reaction was initiated by a 6-fold dilution of the acid-unfolded apo-myoglobin with either 60 mM acetate buffer or 30 mM citrate buffer in D2O delivered from syringe Q, to a final (uncorrected) pH of 5.8. After refolding times (tf) of 0.4 or 6 ms, the H/D exchange reaction was initiated by a 1/6-fold dilution of the sample solution with D2O buffer containing 350-mM 3-(cyclohexylamino)-1 propanesulfonic acid (CAPS) delivered from syringe R at the desired pH* from 7 to 10.7 to enhance exchange of unprotected amide protons to deuterons. The H/D exchange labeling was allowed to proceed for 3.6 ms."

Protection threshold: k(cl)/k(op) > 80

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
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Method: Pulse labeling HDX NMR

Conditions: pH 5.8; 25.0 Celsius; Probes: 51

Related publication:
 PMID 18779573

Experiment details: "A solution containing the fully unfolded apo-myoglobin (80 M) at pH 2.2 in HCl/H2O was placed in syringe P. Refolding and H/D exchange was performed at room temperature. The refolding reaction was initiated by a 6-fold dilution of the acid-unfolded apo-myoglobin with either 60 mM acetate buffer or 30 mM citrate buffer in D2O delivered from syringe Q, to a final (uncorrected) pH of 5.8. After refolding times (tf) of 0.4 or 6 ms, the H/D exchange reaction was initiated by a 1/6-fold dilution of the sample solution with D2O buffer containing 350-mM 3-(cyclohexylamino)-1 propanesulfonic acid (CAPS) delivered from syringe R at the desired pH* from 7 to 10.7 to enhance exchange of unprotected amide protons to deuterons. The H/D exchange labeling was allowed to proceed for 3.6 ms."

Protection threshold: 50 < k(cl)/k(op) < 80

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Pulse labeling HDX NMR

Conditions: pH 5.8; 25.0 Celsius; Probes: 51

Related publication:
 PMID 18779573

Experiment details: "A solution containing the fully unfolded apo-myoglobin (80 M) at pH 2.2 in HCl/H2O was placed in syringe P. Refolding and H/D exchange was performed at room temperature. The refolding reaction was initiated by a 6-fold dilution of the acid-unfolded apo-myoglobin with either 60 mM acetate buffer or 30 mM citrate buffer in D2O delivered from syringe Q, to a final (uncorrected) pH of 5.8. After refolding times (tf) of 0.4 or 6 ms, the H/D exchange reaction was initiated by a 1/6-fold dilution of the sample solution with D2O buffer containing 350-mM 3-(cyclohexylamino)-1 propanesulfonic acid (CAPS) delivered from syringe R at the desired pH* from 7 to 10.7 to enhance exchange of unprotected amide protons to deuterons. The H/D exchange labeling was allowed to proceed for 3.6 ms."

Protection threshold: 20 < k(cl)/k(op) < 50

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Native exchange NMR

Conditions: pH 6.0; 5.0 Celsius; Probes: every amide site

Related publication:
 PMID 2218495

Experiment details: "Apo-Mb in H2O solution was diluted into buffered D2O, initiating H/D exchange. After a period of "exchange-out," further exchange was quenched by the addition of heme and adjusting the pH, and exchange was evaluated by 2D H NMR."

Protection threshold: P > 50000

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Native exchange NMR

Conditions: pH 6.0; 5.0 Celsius; Probes: every amide site

Related publication:
 PMID 2218495

Experiment details: "Apo-Mb in H2O solution was diluted into buffered D2O, initiating H/D exchange. After a period of "exchange-out," further exchange was quenched by the addition of heme and adjusting the pH, and exchange was evaluated by 2D H NMR."

Protection threshold: 5000 < P < 50000

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
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Method: Native exchange in partially folded state by NMR

Conditions: pH 4.2; 5.0 Celsius; Probes: every amide site

Related publication:
 PMID 2218495

Experiment details: "Apo-Mb in H2O solution was diluted into buffered D2O, initiating H/D exchange. After a period of "exchange-out," further exchange was quenched by the addition of heme and adjusting the pH, and exchange was evaluated by 2D H NMR."

Protection threshold: P > 30

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Native exchange in partially folded state by NMR

Conditions: pH 4.2; 5.0 Celsius; Probes: every amide site

Related publication:
 PMID 2218495

Experiment details: "Apo-Mb in H2O solution was diluted into buffered D2O, initiating H/D exchange. After a period of "exchange-out," further exchange was quenched by the addition of heme and adjusting the pH, and exchange was evaluated by 2D H NMR."

Protection threshold: 10 < P < 30

Sequence: VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG
 CLICK TO DOWNLOAD SEQUENCE IN FASTA