Start2Fold

The database of hydrogen/deuterium exchange data on protein folding and stability

Entry STF0026

RNase H domain from HIV-1 reverse transcriptase

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Protein information

Name of the protein: Gag-Pol polyprotein
Organism: Human immunodeficiency virus type 1 group M subtype B (isolate BH10) (HIV-1)
Number of residues: 136
Related UniProt entry:   P03366 (Fragment: 1026 - 1161)
Related PDB entry:   1HRH

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Experiment sets

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EARLY

Method: Pulse labeling HDX NMR

Conditions: pH 5.5; 25.0 Celsius; Probes: 23

Related publication:
 PMID 9792104

Experiment details: "The pulse-labeling hydrogen exchange was carried out in a Bio- Logic SFM4/Q quench flow machine. Continuous flow mode was used for time points at 12, 22, 36, 74, 136, 363, and 636 ms, while the 1, 1.5, and 6 s time points were collected using an interrupted flow technique. Refolding of completely denatured and deuterated RNaseH was initiated by a 1:10 dilution into a protonated folding buffer (20 mM sodium acetate, 50 mM potassium chloride buffer, pHread 5.5). At the specified refolding time points, labeling of amide protons was achieved by a 1:2 dilution with pulse buffer (200 mM tridacetate for a final pHread of 8.0, 8.4, 8.5, 9.0, and also 200 mM glycine for a final PHread of 9.0). After 20 ms, the labeling pulse was quenched by a twofold dilution with 300 mM sodium acetate, pHread 4.5. The sample was immediately put on ice, concentrated in Amicon cells to ~5 mL and the buffer exchanged to 3 mM deuterated sodium acetate in H2O, pHread 4.5."

Protection threshold: P > 10 at 74 ms

Sequence: YQLEKEPIVGAETFYVDGAANRETKLGKAGYVTNKGRQKVVPLTNTTNQKTELQAIYLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKEKVYLAWVPXXXXXGGNEQVDKLVSAGI
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EARLY residues

15: Y; 54: Q; 55: A; 57: Y; 59: A; 68: N; 69: I; 94: Q; 95: I; 98: Q; 99: L; 106: Y; 108: A;
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INTERMEDIATE

Method: Pulse labeling HDX NMR

Conditions: pH 5.5; 25.0 Celsius; Probes: 23

Related publication:
 PMID 9792104

Experiment details: "The pulse-labeling hydrogen exchange was carried out in a Bio- Logic SFM4/Q quench flow machine. Continuous flow mode was used for time points at 12, 22, 36, 74, 136, 363, and 636 ms, while the 1, 1.5, and 6 s time points were collected using an interrupted flow technique. Refolding of completely denatured and deuterated RNaseH was initiated by a 1:10 dilution into a protonated folding buffer (20 mM sodium acetate, 50 mM potassium chloride buffer, pHread 5.5). At the specified refolding time points, labeling of amide protons was achieved by a 1:2 dilution with pulse buffer (200 mM tridacetate for a final pHread of 8.0, 8.4, 8.5, 9.0, and also 200 mM glycine for a final PHread of 9.0). After 20 ms, the labeling pulse was quenched by a twofold dilution with 300 mM sodium acetate, pHread 4.5. The sample was immediately put on ice, concentrated in Amicon cells to ~5 mL and the buffer exchanged to 3 mM deuterated sodium acetate in H2O, pHread 4.5."

Protection threshold: 3 < P < 10 at 74 ms

Sequence: YQLEKEPIVGAETFYVDGAANRETKLGKAGYVTNKGRQKVVPLTNTTNQKTELQAIYLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKEKVYLAWVPXXXXXGGNEQVDKLVSAGI
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INTERMEDIATE residues

53: L; 56: I; 58: L; 60: L; 97: E;
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STRONG

Method: Native exchange NMR

Conditions: pH 5.5; 25.0 Celsius; Probes: 112

Related publication:
 PMID 9792104

Experiment details: "None"

Protection threshold: exchange less than 5% during the sample preparation and NMR data collection

Sequence: YQLEKEPIVGAETFYVDGAANRETKLGKAGYVTNKGRQKVVPLTNTTNQKTELQAIYLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKEKVYLAWVPXXXXXGGNEQVDKLVSAGI
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

STRONG residues

15: Y; 16: V; 53: L; 54: Q; 55: A; 56: I; 57: Y; 58: L; 59: A; 60: L; 67: V; 68: N; 69: I; 70: V; 94: Q; 95: I; 97: E; 98: Q; 99: L; 100: I; 106: Y; 108: A;
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