Entry STF0031
Human alpha1-antitrypsin
Protein information
Name of the protein: | Alpha-1-antitrypsin |
Organism: | Homo sapiens |
Number of residues: | 394 |
Related UniProt entry: | P01009 (Fragment: 47 - 440) |
Related PDB entry: | 1QLP |
Visualize the data
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Experiment sets
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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