Entry STF0011
E. coli dihydrofolate reductase (DHFR)
Protein information
Name of the protein: | Dihydrofolate reductase |
Organism: | Escherichia coli (strain K12) |
Number of residues: | 159 |
Related UniProt entry: | P0ABQ4 (Fragment: 1 - 159) |
Related PDB entry: | 5DFR |
Visualize the data
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Experiment sets
EARLY
Method: Pulse labeling HDX NMR
Conditions: pH 6.3; 15.0 Celsius; Probes: 26
Related publication:
PMID 7757007
Experiment details: "Pulse labeling experiments were performed in a QFM-5 rapid-mixing device. DHFR (3 mg/mL) was denatured and all exchangeable amide hydrogens were deuterated by incubation in 6 M D4-urea, 50 mM potassium phosphate in 99.9% D2O (pH 6.3) at 15 °C for 1 h. Refolding was initiated by rapid sixfold dilution in 50 mM potassium phosphate buffer, pH* 6.3, containing 0.2 mM K2EDTA and 1 mM P-mercaptoethanol for variable time periods (t,) prior to the labeling pulse. The labeling pulse was initiated by a twofold dilution into either 200 mM tris-HCI for pulse pH* values below 9.5, or 200 mM glycine for pH* values at and above 9.5; both buffers contained 0.2 mM K2EDTA and 1 mM 6-mercaptoethanol."
Protection threshold: Strongly protected within 13 ms
Sequence:
MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
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EARLY residues
81: A;
92: M;
103: F;
111: Y;
157: E;
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INTERMEDIATE
Method: Pulse labeling HDX NMR
Conditions: pH 6.3; 15.0 Celsius; Probes: 26
Related publication:
PMID 7757007
Experiment details: "Pulse labeling experiments were performed in a QFM-5 rapid-mixing device. DHFR (3 mg/mL) was denatured and all exchangeable amide hydrogens were deuterated by incubation in 6 M D4-urea, 50 mM potassium phosphate in 99.9% D2O (pH 6.3) at 15 °C for 1 h. Refolding was initiated by rapid sixfold dilution in 50 mM potassium phosphate buffer, pH* 6.3, containing 0.2 mM K2EDTA and 1 mM P-mercaptoethanol for variable time periods (t,) prior to the labeling pulse. The labeling pulse was initiated by a twofold dilution into either 200 mM tris-HCI for pulse pH* values below 9.5, or 200 mM glycine for pH* values at and above 9.5; both buffers contained 0.2 mM K2EDTA and 1 mM 6-mercaptoethanol."
Protection threshold: Weakly protected within 13 ms
Sequence:
MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
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INTERMEDIATE residues
5: I;
40: V;
41: I;
75: V;
91: I;
94: I;
155: I;
156: L;
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LATE
Method: Pulse labeling HDX NMR
Conditions: pH 6.3; 15.0 Celsius; Probes: 26
Related publication:
PMID 7757007
Experiment details: "Pulse labeling experiments were performed in a QFM-5 rapid-mixing device. DHFR (3 mg/mL) was denatured and all exchangeable amide hydrogens were deuterated by incubation in 6 M D4-urea, 50 mM potassium phosphate in 99.9% D2O (pH 6.3) at 15 °C for 1 h. Refolding was initiated by rapid sixfold dilution in 50 mM potassium phosphate buffer, pH* 6.3, containing 0.2 mM K2EDTA and 1 mM P-mercaptoethanol for variable time periods (t,) prior to the labeling pulse. The labeling pulse was initiated by a twofold dilution into either 200 mM tris-HCI for pulse pH* values below 9.5, or 200 mM glycine for pH* values at and above 9.5; both buffers contained 0.2 mM K2EDTA and 1 mM 6-mercaptoethanol."
Protection threshold: Not protected within 13 ms
Sequence:
MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
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LATE residues
3: S;
4: L;
42: M;
60: I;
109: K;
110: L;
112: L;
113: T;
114: H;
139: E;
151: Y;
153: F;
154: E;
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STRONG
Method: Native exchange NMR
Conditions: pH 6.86; 25.0 Celsius; Probes: all
Related publication:
PMID 7757007
Experiment details: "Exchange-out experiment at pH* 6.86 and 25 °C. A lyophilized, fully protonated sample of DHFR containing 1.5 molar excess of folate and appropriate buffer components was dissolved in D2O and a 2QF-COSY spectrum was recorded immediately. Following the acquisition of the initial 2QF-COSY spectrum (- 12 h), a second spectrum was recorded. Cross peaks for amide hydrogens that did not decrease in intensity by more than 25% in the second spectrum relative to the first were chosen."
Protection threshold: amids persisting to exchange for 12 hours
Sequence:
MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR
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STRONG residues
3: S;
4: L;
5: I;
40: V;
41: I;
42: M;
60: I;
75: V;
81: A;
91: I;
92: M;
94: I;
103: F;
109: K;
110: L;
111: Y;
112: L;
113: T;
114: H;
139: E;
151: Y;
153: F;
154: E;
155: I;
156: L;
157: E;
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