Native exchange in partially folded state by NMR None P > 30 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.

Pulse labeling HDX NMR None k(cl)/k(op) > 80 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.

Pulse labeling HDX NMR None 50 < k(cl)/k(op) < 80 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.

Pulse labeling HDX NMR None 20 < k(cl)/k(op) < 50 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.

Native exchange NMR None P > 50000 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.

Native exchange NMR None 5000 < P < 50000 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.

Native exchange in partially folded state by NMR None 10 < P < 30 VLSEGEWQLVLHVWAKVEADVAGHGQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASEDLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKELGYQG

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.