Method: Pulse labeling HDX MS

Conditions: pH 5.0; 10.0 Celsius; Probes: every amide site

Related publication:
 PMID 23603271

Experiment details: "RNase H* is a mutant C13A C63A C133A of E. coli RNase H. Denaturant-unfolded protein in D2O is diluted into H2O folding buffer under conditions in which D-to-H back exchange is very slow (pH 5.0, 10 C;~40-s HX time constant). After trial folding times, a brief labeling pulse to high pH is imposed (10 ms, pH 10, 10 °C), equivalent to 25 times the average HX time constant. The subsequent HX MS analysis is designed to detect which residues are protected at each folding time."

Protection threshold: > 50% protection in competition phase (< 1 ms folding time)

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
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Method: Pulse labeling HDX MS

Conditions: pH 5.0; 10.0 Celsius; Probes: every amide site

Related publication:
 PMID 23603271

Experiment details: "RNase H* is a mutant C13A C63A C133A of E. coli RNase H. Denaturant-unfolded protein in D2O is diluted into H2O folding buffer under conditions in which D-to-H back exchange is very slow (pH 5.0, 10 C;~40-s HX time constant). After trial folding times, a brief labeling pulse to high pH is imposed (10 ms, pH 10, 10 °C), equivalent to 25 times the average HX time constant. The subsequent HX MS analysis is designed to detect which residues are protected at each folding time."

Protection threshold: > 50% protection in competition phase (< 9 ms folding time)

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Pulse labeling HDX MS

Conditions: pH 5.0; 10.0 Celsius; Probes: every amide site

Related publication:
 PMID 23603271

Experiment details: "RNase H* is a mutant C13A C63A C133A of E. coli RNase H. Denaturant-unfolded protein in D2O is diluted into H2O folding buffer under conditions in which D-to-H back exchange is very slow (pH 5.0, 10 C;~40-s HX time constant). After trial folding times, a brief labeling pulse to high pH is imposed (10 ms, pH 10, 10 °C), equivalent to 25 times the average HX time constant. The subsequent HX MS analysis is designed to detect which residues are protected at each folding time."

Protection threshold: > 50% protection in competition phase (< 720 ms folding time)

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
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Method: Native exchange NMR

Conditions: pH 5.1; 25.0 Celsius; Probes: -

Related publication:
 PMID 8784352

Experiment details: "None"

Protection threshold: slowest exchange

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
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Method: Native exchange NMR

Conditions: pH 5.5; 27.0 Celsius; Probes: 61

Related publication:
 PMID 9533889

Experiment details: "A series of 2D1H ±15N HSQC spectra were obtained on the 600 MHz spectrometer, which was initiated 30 minutes after the solubilization of the sample in the D2O buffer. The first eight HSQC spectra, with measurement times of 30 minutes each, were successively obtained (starting times of the measurements after the solubilization were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 hours). Other HSQC spectra, with measurement times of 2.0 hours, were obtained with starting times of 4.5, 6.5, 8.5, 10.5, 14, 20, 40, 66, 91, 164, 238, 328.5, 495, 666, 933, 1652, 2758, 3089, and 3492 hours for the wild-type RNase HI."

Protection threshold: k(ex)/min < 2.56E–07

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
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Method: HDX-folding competition by NMR

Conditions: pH 5.5-7.5; 25.0 Celsius; Probes: 59

Related publication:
 PMID 8527428

Experiment details: "The competition method was applied at three different pH values (5.5, 6.5, and 7.5) to adjust the hydrogen-exchange rate. For pH 5.5, 10 mM acetate buffer from CD3COOD (CIL) NaOH used. For pH 7.5 and 6.10 mM phosphate buffer was used. The buffers made with D2O were designated as D2O buffers to distinguish them from the buffers made with H2O (H2O buffers). For the D2O buffers, the pH values were not corrected for isotope effects. Four milligrams of RNase HI was dissolved in 200 μl of the H2O buffers containing 7.0 M urea. This protein solution (200 μl) and the D2O buffer (5 mL), at the corresponding pDread without urea, were preincubated at 25 °C for more than 5 min. The competition between the refolding and the H-D exchange reactions was initiated by mixing these two solutions in a very short time (within 500 ms). After 1 min, the H-D exchange reaction was quenched by ice cooling and pH adjustment by dilution with 10 mL of a D2O buffer [250 mM CD3COONa (pDread 5.5)]."

Protection threshold: relative cross-peak intensities more than 0.7

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: HDX-folding competition by NMR

Conditions: pH 5.5-7.5; 25.0 Celsius; Probes: 59

Related publication:
 PMID 8527428

Experiment details: "The competition method was applied at three different pH values (5.5, 6.5, and 7.5) to adjust the hydrogen-exchange rate. For pH 5.5, 10 mM acetate buffer from CD3COOD (CIL) NaOH used. For pH 7.5 and 6.10 mM phosphate buffer was used. The buffers made with D2O were designated as D2O buffers to distinguish them from the buffers made with H2O (H2O buffers). For the D2O buffers, the pH values were not corrected for isotope effects. Four milligrams of RNase HI was dissolved in 200 μl of the H2O buffers containing 7.0 M urea. This protein solution (200 μl) and the D2O buffer (5 mL), at the corresponding pDread without urea, were preincubated at 25 °C for more than 5 min. The competition between the refolding and the H-D exchange reactions was initiated by mixing these two solutions in a very short time (within 500 ms). After 1 min, the H-D exchange reaction was quenched by ice cooling and pH adjustment by dilution with 10 mL of a D2O buffer [250 mM CD3COONa (pDread 5.5)]."

Protection threshold: relative cross-peak intensities between 0.5 and 0.7

Sequence: MLKQVEIFTDGSALGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHAEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERADELARAAAMNPTLEDTGYQVEV
 CLICK TO DOWNLOAD SEQUENCE IN FASTA