Start2Fold

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

Entry STF0030

Apo-flavodoxin

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Protein information

Name of the protein: Flavodoxin-2
Organism: Borrelia hermsii (strain HS1 / DAH)
Number of residues: 179
Related UniProt entry:   P00324 (Fragment: 2 - 180)
Related PDB entry:   1YOB

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Experiment sets

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EARLY

Method: Denaturant-dependent disappearance of amide cross peaks by NMR

Conditions: pH 6.0; 25.0 Celsius; Probes: 114

Related publication:
 PMID 19170491

Experiment details: "Unfolded apoflavodoxin was obtained by denaturing flavodoxin in 6 M guanidine hydrochloride. Subsequently, FMN was removed via gel filtration at 7 M GuHCl. 550 μL NMR samples of apoflavodoxin at various denaturant concentrations were prepared; in total, 18 samples with GuHCl concentrations ranging from 4.05 to 1.58 M GuHCl. The buffer used in all experiments was 100 mM potassium pyrophosphate, pH 6.0. 1H-15N HSQC spectra of unfolded apoflavodoxin were acquired. Subsequently, the chemical shifts of the amides of the unfolded protein were determined in these spectra. In addition, the denaturant-dependent disappearance of the amide cross peaks of unfolded apoflavodoxin were tracked."

Protection threshold: 2.5 M GuHCl < Cm (midpoint of folding) < 2.8 M GuHCl

Sequence: AKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDALNVNRVSAEDFAQYQFLILGTPTLGEGELPGLSSDAENESWEEFLPKIEGLDFSGKTVALFGLGDQVGYPENYLDALGELYSFFKDRGAKIVGSWSTDGYEFESSEAVVDGKFVGLALDLDNQSGKTDERVAAWLAQIAPEFGLSL
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EARLY residues

52: L; 53: G; 78: L; 82: E; 87: S; 92: A; 109: A; 111: G; 115: S; 126: G; 127: S; 148: G; 165: A; 168: L; 169: A; 170: Q; 171: I; 172: A;
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INTERMEDIATE

Method: Denaturant-dependent disappearance of amide cross peaks by NMR

Conditions: pH 6.0; 25.0 Celsius; Probes: 114

Related publication:
 PMID 19170491

Experiment details: "Unfolded apoflavodoxin was obtained by denaturing flavodoxin in 6 M guanidine hydrochloride. Subsequently, FMN was removed via gel filtration at 7 M GuHCl. 550 μL NMR samples of apoflavodoxin at various denaturant concentrations were prepared; in total, 18 samples with GuHCl concentrations ranging from 4.05 to 1.58 M GuHCl. The buffer used in all experiments was 100 mM potassium pyrophosphate, pH 6.0. 1H-15N HSQC spectra of unfolded apoflavodoxin were acquired. Subsequently, the chemical shifts of the amides of the unfolded protein were determined in these spectra. In addition, the denaturant-dependent disappearance of the amide cross peaks of unfolded apoflavodoxin were tracked."

Protection threshold: 2.2 M GuHCl < Cm (midpoint of folding) < 2.5 M GuHCl

Sequence: AKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDALNVNRVSAEDFAQYQFLILGTPTLGEGELPGLSSDAENESWEEFLPKIEGLDFSGKTVALFGLGDQVGYPENYLDALGELYSFFKDRGAKIVGSWSTDGYEFESSEAVVDGKFVGLALDLDNQSGKTDERVAAWLAQIAPEFGLSL
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INTERMEDIATE residues

46: Q; 54: T; 83: G; 86: F; 95: G; 101: G; 122: A; 129: S; 130: T; 143: D; 144: G; 150: A; 167: W; 176: G;
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STRONG

Method: Native exchange NMR

Conditions: pH 6.2; 30.0 Celsius; Probes: 57+2

Related publication:
 PMID 9737928

Experiment details: "The H/D exchange measurements were carried out on the resulting 2 mM 15N-labeled apoflavodoxin sample over a five-day period (pH* 6.2, 303 K). The first gradient-enhanced 1H-15N HSQC experiment was started 12 minutes after initiation of exchange and each experiment lasted eight minutes. Peak intensities were measured and used to calculate the amide proton exchange rate, k(ex). ΔG(op) values were determined from the observed rate constants, k(ex), and the intrinsic chemical exchange rates, k(int)."

Protection threshold: ΔG(op) ≥ 6.2 kcal/mol

Sequence: AKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDALNVNRVSAEDFAQYQFLILGTPTLGEGELPGLSSDAENESWEEFLPKIEGLDFSGKTVALFGLGDQVGYPENYLDALGELYSFFKDRGAKIVGSWSTDGYEFESSEAVVDGKFVGLALDLDNQSGKTDERVAAWLAQIAPEFGLSL
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

STRONG residues

5: L; 6: F; 49: F; 50: L; 51: I; 52: L; 53: G; 81: I; 90: T; 91: V; 92: A; 93: L; 94: F; 111: G; 114: Y; 117: F; 118: K; 120: R; 123: K; 125: V; 165: A; 167: W; 168: L; 169: A; 170: Q; 171: I; 172: A;
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