Start2Fold

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

Entry STF0008

Cobrotoxin (CBTX)

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Protein information

Name of the protein: Cobrotoxin
Organism: Naja atra (Chinese cobra)
Number of residues: 62
Related UniProt entry:   P60770 (Fragment: 22 - 83)
Related PDB entry:   1COE

Visualize the data

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Experiment sets

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EARLY

Method: Quenched-flow HDX NMR

Conditions: pH 3.0; 5.0 Celsius; Probes: 24

Related publication:
 PMID 16497267

Experiment details: "All experiments were carried out at 5C using a RQF-63 rapid mixing quenched-flow apparatus. Complete denaturation and exchange of the backbone amide protons with deuterium was achieved by dissolving CBTX (20 mg/mL) in 6 M urea-d4 in D2O at pD 2.7. Refolding of denatured protein was initiated by a 11-fold dilution with 50 mM glycine-d5 (pH 3.0) in H2O. At this pH, H/D exchange rate was negligible. After variable refolding times ranging from 9.8 to 500 ms, the solution was diluted again to 11 times of the initial protein volume with 0.2 M sodium borate (pH 9.5) to initiate labeling of the deuterated amides in CBTX with protons."

Protection threshold: refolding time constant < 20

Sequence: LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN
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EARLY residues

28: R; 45: S; 50: I; 53: N; 55: C; 62: N;
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INTERMEDIATE

Method: Quenched-flow HDX NMR

Conditions: pH 3.0; 5.0 Celsius; Probes: 24

Related publication:
 PMID 16497267

Experiment details: "All experiments were carried out at 5C using a RQF-63 rapid mixing quenched-flow apparatus. Complete denaturation and exchange of the backbone amide protons with deuterium was achieved by dissolving CBTX (20 mg/mL) in 6 M urea-d4 in D2O at pD 2.7. Refolding of denatured protein was initiated by a 11-fold dilution with 50 mM glycine-d5 (pH 3.0) in H2O. At this pH, H/D exchange rate was negligible. After variable refolding times ranging from 9.8 to 500 ms, the solution was diluted again to 11 times of the initial protein volume with 0.2 M sodium borate (pH 9.5) to initiate labeling of the deuterated amides in CBTX with protons."

Protection threshold: 20 < refolding time constant < 40

Sequence: LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

INTERMEDIATE residues

6: Q; 14: T; 15: T; 26: K; 27: K; 33: R;
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LATE

Method: Quenched-flow HDX NMR

Conditions: pH 3.0; 5.0 Celsius; Probes: 24

Related publication:
 PMID 16497267

Experiment details: "All experiments were carried out at 5C using a RQF-63 rapid mixing quenched-flow apparatus. Complete denaturation and exchange of the backbone amide protons with deuterium was achieved by dissolving CBTX (20 mg/mL) in 6 M urea-d4 in D2O at pD 2.7. Refolding of denatured protein was initiated by a 11-fold dilution with 50 mM glycine-d5 (pH 3.0) in H2O. At this pH, H/D exchange rate was negligible. After variable refolding times ranging from 9.8 to 500 ms, the solution was diluted again to 11 times of the initial protein volume with 0.2 M sodium borate (pH 9.5) to initiate labeling of the deuterated amides in CBTX with protons."

Protection threshold: 40 < refolding time constant < 60

Sequence: LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

LATE residues

2: E; 10: Q; 17: C; 24: C; 25: Y; 29: W; 30: R; 58: D; 61: N;
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STRONG

Method: Native exchange NMR

Conditions: pH 3.4; 25.0 Celsius; Probes: 38

Related publication:
 PMID 10913281

Experiment details: "H/D exchange measurements in CTX III and CBTX were monitored using the magnitude COSY spectra recorded at 25°C (pH 3.4) using a Bruker DMX-600 NMR spectrometer. The samples for exchange kinetics of the amide protons in the proteins (CBTX and CTX III) were prepared by dissolving the lyophilized proteins in deuterated buffer at pD 3.6. The concentrations of the proteins (CBTX and CTX III) were 2.0 mM."

Protection threshold: log(P) > 2

Sequence: LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN
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STRONG residues

3: C; 6: Q; 9: S; 14: T; 15: T; 24: C; 25: Y; 26: K; 27: K; 29: W; 35: Y; 38: E; 39: R; 42: G; 53: N; 55: C; 59: R;
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MEDIUM

Method: Native exchange NMR

Conditions: pH 3.4; 25.0 Celsius; Probes: 38

Related publication:
 PMID 10913281

Experiment details: "H/D exchange measurements in CTX III and CBTX were monitored using the magnitude COSY spectra recorded at 25°C (pH 3.4) using a Bruker DMX-600 NMR spectrometer. The samples for exchange kinetics of the amide protons in the proteins (CBTX and CTX III) were prepared by dissolving the lyophilized proteins in deuterated buffer at pD 3.6. The concentrations of the proteins (CBTX and CTX III) were 2.0 mM."

Protection threshold: 1 < log(P) < 2

Sequence: LECHNQQSSQTPTTTGCSGGETNCYKKRWRDHRGYRTERGCGCPSVKNGIEINCCTTDRCNN
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

MEDIUM residues

2: E; 17: C; 21: E; 28: R; 30: R; 37: T; 41: C; 48: N; 50: I; 51: E; 52: I;
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