Start2Fold

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

Entry STF0031

Human alpha1-antitrypsin

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Protein information

Name of the protein: Alpha-1-antitrypsin
Organism: Bos taurus (Bovine)
Number of residues: 394
Related UniProt entry:   P01009 (Fragment: 47 - 440)
Related PDB entry:   1QLP

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Experiment sets

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EARLY

Method: Oxidative labeling MS

Conditions: pH 7.8; 22.0 Celsius; Probes: 33 peptides covering 93% of the sequence

Related publication:
 PMID 22940366

Experiment details: "α1AT was initially unfolded in 6 M GdnHCl at 22 °C for 2 hours (pH 7.8). Kinetic folding experiments with oxidative labeling were performed using a two-syringe continuous-flow mixing device. For the 0.5 s and 7 s time points, syringes 1 and 2 were advanced at 2.5 and 47.5 μl/min, respectively, using a syringe pump. Syringe 1 contained 200 μM α1AT denatured in 6 M GdnHCl, 10 mM phosphate buffer (pH 7.8), and 50 mM NaCl. Protein folding was triggered by combining this solution with phosphate buffer (pH 7.8) from syringe 2 at a homemade capillary mixer in a 1:19 volume ratio, resulting in a final denaturant concentration of 0.3 M (far below the unfolding midpoint) and a protein conc. of 10 μM. The outlet of the mixer was connected to a reaction capillary with an internal diameter of 100 μm. Syringe 2 also contained 15 mM glutamine and 0.1% (v/v) (ca 30 mM) H2O2. A KrF excimer laser (GAM EX 50, Orlando, FL) producing 18 ns pulses at 248nm, 72Hz, and 37mJ and an irradiation spot width of ca 1mm was used to generate -OH by photolysis of H2O2 within the reaction capillary. Glutamine acts as a radical scavenger that quenches the labeling reaction on a time scale of 1μs. Following initiation of folding, oxidative labeling was performed by irradiating the reaction mixture at different positions downstream of the mixer. Average reaction times of 0.5 s and 7 s correspond to distances between mixer and irradiation spot of 5.3 cm and 74.2 cm, respectively."

Protection threshold: completely refolded in 10 minutes

Sequence: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
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EARLY residues

1: E; 2: D; 3: P; 4: Q; 5: G; 6: D; 7: A; 8: A; 9: Q; 10: K; 53: S; 54: P; 55: V; 56: S; 57: I; 58: A; 59: T; 60: A; 61: F; 62: A; 63: M; 64: L; 65: S; 66: L; 67: G; 68: T; 69: K; 70: A; 71: D; 72: T; 73: H; 74: D; 75: E; 76: I; 77: L; 104: N; 105: Q; 106: P; 107: D; 108: S; 109: Q; 110: L; 218: V; 219: P; 220: M; 221: M; 222: K;
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LATE

Method: Oxidative labeling MS

Conditions: pH 7.8; 22.0 Celsius; Probes: 33 peptides covering 93% of the sequence

Related publication:
 PMID 22940366

Experiment details: "α1AT was initially unfolded in 6 M GdnHCl at 22 °C for 2 hours (pH 7.8). Kinetic folding experiments with oxidative labeling were performed using a two-syringe continuous-flow mixing device. For the 0.5 s and 7 s time points, syringes 1 and 2 were advanced at 2.5 and 47.5 μl/min, respectively, using a syringe pump. Syringe 1 contained 200 μM α1AT denatured in 6 M GdnHCl, 10 mM phosphate buffer (pH 7.8), and 50 mM NaCl. Protein folding was triggered by combining this solution with phosphate buffer (pH 7.8) from syringe 2 at a homemade capillary mixer in a 1:19 volume ratio, resulting in a final denaturant concentration of 0.3 M (far below the unfolding midpoint) and a protein conc. of 10 μM. The outlet of the mixer was connected to a reaction capillary with an internal diameter of 100 μm. Syringe 2 also contained 15 mM glutamine and 0.1% (v/v) (ca 30 mM) H2O2. A KrF excimer laser (GAM EX 50, Orlando, FL) producing 18 ns pulses at 248nm, 72Hz, and 37mJ and an irradiation spot width of ca 1mm was used to generate -OH by photolysis of H2O2 within the reaction capillary. Glutamine acts as a radical scavenger that quenches the labeling reaction on a time scale of 1μs. Following initiation of folding, oxidative labeling was performed by irradiating the reaction mixture at different positions downstream of the mixer. Average reaction times of 0.5 s and 7 s correspond to distances between mixer and irradiation spot of 5.3 cm and 74.2 cm, respectively."

Protection threshold: refolded in 10-30 minutes

Sequence: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
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LATE residues

26: I; 27: T; 28: P; 29: N; 30: L; 31: A; 32: E; 33: F; 34: A; 35: F; 36: S; 37: L; 38: Y; 39: R; 40: Q; 41: L; 42: A; 43: H; 44: Q; 45: S; 46: N; 47: S; 48: T; 49: N; 50: I; 51: F; 83: N; 84: L; 85: T; 86: E; 87: I; 88: P; 89: E; 90: A; 91: Q; 92: I; 93: H; 94: E; 95: G; 96: F; 113: T; 114: T; 115: G; 116: N; 117: G; 118: L; 119: F; 120: L; 121: S; 122: E; 123: G; 124: L; 125: K; 126: L; 127: V; 128: D; 129: K; 130: F; 131: L; 132: E; 133: D; 134: V; 135: K; 224: L; 225: G; 226: M; 227: F; 228: N; 229: I; 230: Q; 231: H; 232: C; 233: K; 235: L; 236: S; 237: S; 238: W; 239: V; 240: L; 241: L; 242: M; 243: K; 260: L; 261: Q; 262: H; 263: L; 264: E; 265: N; 266: E; 267: L; 268: T; 269: H; 270: D; 271: I; 272: I; 273: T; 274: K; 275: F; 276: L; 277: E; 278: N; 279: E; 280: D; 281: R; 366: F; 367: N; 368: K; 369: P; 370: F; 371: V; 372: F; 373: L; 374: M; 375: I; 376: E; 377: Q; 378: N; 379: T; 380: K; 381: S; 382: P; 383: L; 384: F; 385: M; 386: G; 387: K;
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EARLY

Method: Pulse labeling HDX MS

Conditions: pH 7.8; 22.0 Celsius; Probes: monitors MS segments almost covering the full protein

Related publication:
 PMID 22392975

Experiment details: "To unfold the purified α1AT, 5 μg of the protein was incubated in 10 μL of 10 mM sodium phosphate (pH 7.8) and 50 mM NaCl containing 6 M GuHCl for 2 h at room temperature. The sample was diluted 10-fold with 10 mM sodium phosphate (pH 7.8) and 50 mM NaCl and refolded for increasing amounts of time. At various time points, refolding samples was deuterated for 10 s by an addition of 10 mM sodium phosphate (pD 7.8), 50 mM NaCl containing 0.6 M guanidine deuterochloride to a final volume of 1 mL. The deuteration reaction was quenched by adding HCl to lower pH to 2.3, and the sample was frozen and stored at −80°C until use."

Protection threshold: faster protection (without lag phase)

Sequence: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
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EARLY residues

38: Y; 39: R; 40: Q; 41: L; 42: A; 43: H; 44: Q; 45: S; 46: N; 47: S; 48: T; 49: N; 50: I; 51: F; 52: F; 53: S; 54: P; 55: V; 56: S; 57: I; 58: A; 59: T; 60: A; 120: L; 121: S; 122: E; 123: G; 124: L; 125: K; 126: L; 127: V; 128: D; 129: K; 130: F; 131: L; 132: E; 133: D; 134: V; 135: K; 136: K; 137: L; 138: Y; 139: H; 140: S; 141: E; 142: A; 209: H; 210: V; 211: D; 212: Q; 213: V; 214: T; 215: T; 216: V; 217: K; 218: V; 219: P; 220: M; 221: M; 222: K; 223: R; 224: L; 225: G; 226: M; 227: F; 251: I; 252: F; 253: F; 254: L; 255: P; 256: D; 257: E; 258: G; 259: K; 260: L; 261: Q; 262: H; 263: L; 264: E; 265: N; 266: E; 267: L; 268: T; 269: H; 270: D; 271: I; 272: I; 273: T; 274: K; 275: F;
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