Two-fold symmetric structure of the 18-60 construct of S31N M2 from Influenza A in lipid bilayers
Polymer type: polypeptide(L)
Total | 1H | 13C | 15N | |
---|---|---|---|---|
All | 43.4 % (235 of 542) | 19.7 % (55 of 279) | 67.9 % (148 of 218) | 71.1 % (32 of 45) |
Backbone | 58.2 % (149 of 256) | 34.5 % (30 of 87) | 70.1 % (89 of 127) | 71.4 % (30 of 42) |
Sidechain | 35.2 % (115 of 327) | 13.0 % (25 of 192) | 66.7 % (88 of 132) | 66.7 % (2 of 3) |
Aromatic | 19.1 % (13 of 68) | 2.9 % (1 of 34) | 33.3 % (11 of 33) | 100.0 % (1 of 1) |
Methyl | 56.3 % (36 of 64) | 18.8 % (6 of 32) | 93.8 % (30 of 32) |
1. matrix protein 2
RSNDSSDPLV VAANIIGILH LILWILDRLF FKSIYRFFEH GLKSolvent system H2O, Temperature 298 (±5) K, pH 7.8, Details Per liter, 3 g 13C glucose and 1 g 15N ammonium chloride (CIL), with another 0.5 to 1 g 13C glucose added at induction (150 M IPTG, 18 C) at an OD600 of 0.7 to 0.9. The final OD600 reached 3.5 to 4, after 18 hours of expression at 18 C, 15mg of pure protein.
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | sodium phosphate | natural abundance | buffer | 40 (±1.0) mM |
2 | glutamic acid | natural abundance | 30 (±1.0) mM | |
3 | sodium azide | natural abundance | 3 (±1.0) mM | |
4 | 1,2-diphytanoyl-sn-glycero-3-phosphocholine | natural abundance | 50 % w/w | |
5 | matrix protein 2 | [U-13C,15N] | protein | 50 % w/w |
6 | H2O | natural abundance | solvent | 100 % |
Solvent system H2O, Temperature 298 (±5) K, pH 7.8, Details Per liter, 3 g 13C glucose and 1 g 15N ammonium chloride (CIL), with another 0.5 to 1 g 13C glucose added at induction. Cells were grown in 700 mL of media, with 300 mL of media used to dissolve the amino acids. When the OD600 reached 0.7, the reserved 300 mL of media was added, along with amino acids at natural isotopic abundance:23,24 150 mg isoleucine, 150 mg leucine, 50 mg phenylalanine, and 100 mg tyrosine (Sigma-Aldrich). Expression of protein was induced with 150 M IPTG at 18 C as before. The final OD600 reached 3.5 to 4.6, after 18 hours of expression at 18 C, and yielded up to 15 mg of pure protein.
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
7 | sodium phosphate | natural abundance | buffer | 40 (±1.0) mM |
8 | glutamic acid | natural abundance | 30 (±1.0) mM | |
9 | sodium azide | natural abundance | 3 (±1.0) mM | |
10 | 1,2-diphytanoyl-sn-glycero-3-phosphocholine | natural abundance | 50 % w/w | |
11 | matrix protein 2 | U-13C,15N-[12C,14N-ILFY] | protein | 50 % w/w |
12 | H2O | natural abundance | solvent | 100 % |
Solvent system H2O, Temperature 298 (±5) K, pH 7.8, Details Preparation was identical to U-13C-15N M2 except that specifically isotopically labeled glucose was used. The labeling pattern resulting from [1,6-13C]-glucose is the same as from [1-13C]-glucose25 (Omicron Biochemicals) except that the [1,6-13C]-glucose results in double the labeling level per site.
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
19 | sodium phosphate | natural abundance | buffer | 40 (±1.0) mM |
20 | glutamic acid | natural abundance | 30 (±1.0) mM | |
21 | sodium azide | natural abundance | 3 (±1.0) mM | |
22 | 1,2-diphytanoyl-sn-glycero-3-phosphocholine | natural abundance | 50 % w/w | |
23 | matrix protein 2 | [1,6-13C2]-Glucose | protein | 50 % w/w |
24 | H2O | natural abundance | solvent | 100 % |
Solvent system H2O, Temperature 298 (±5) K, pH 7.8, Details An overnight culture in 50 mL of 90 % 2H M9, 10 % SOC media was grown from a fresh transformation in BL21-DE3 until an OD600 of 2 to 3. Cells were then pelleted by centrifugation and transferred to 1L of 2H M9 media containing 3 g 2H-13C glucose, 1 g 15N ammonium chloride, salts, and Centrum in 1 L of 99.8 % 2H D2O. The doubling time was 2 hours. At an OD600 of 0.65 to 0.75, ILV precursors were added: 75 mg alpha-keto-butyric acid,26 sodium salt 4-13C 99 %, 3,3,4,4-2H4, 98 % (CIL), and 350 mg of 2-(13C,2H2)methyl-4-(2H3)-acetolactate prepared as described previously. Cells were harvested after 24 to 36 hours, and yielded up to 5 mg of pure protein. This labeling pattern re-sults in methyl groups with isolated 13C1H spin pairs in an otherwise 2H, 12C background at nearby sites. The protein was purified and refolded in 1H buffers, resulting in complete exchange to 1H at exchangeable sites such as the backbone amides.
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
25 | sodium phosphate | natural abundance | buffer | 40 (±1.0) mM |
26 | glutamic acid | natural abundance | 30 (±1.0) mM | |
27 | sodium azide | natural abundance | 3 (±1.0) mM | |
28 | 1,2-diphytanoyl-sn-glycero-3-phosphocholine | aliphatic chain [U-2H] | 50 % w/w | |
29 | matrix protein 2 | U-13C,15N,2H-[12C,13C2H21H 1-Ile, 12C,13Ca,13C',13C2H21H 2-Leu, 12C,13C2H21H 2-Val] | protein | 50 % w/w |
30 | H2O | natural abundance | solvent | 100 % |