Introduction
Methods
Chemicals
CBS mutants
Responsivity of mutants to chemical chaperones
Toxicity of chemical chaperones for E. coli
Intracellular chaperones concentrations
Expression in E. coli
Specific activity of mutants
Native polyacrylamide gel electrophoresis and Western blot
Results
Transport of chaperones into E. coli
Chaperones and ligands in SOB medium | Chaperones and ligands in E. coli, mmol/kg wet weight | ||
---|---|---|---|
- Addition | + Addition | ||
Chaperones | Glycerol 750 mmol/l | 2.3 | 64.6 |
Betaine 100 mmol/l | 0.4 | 11.0 | |
Taurine 50 mmol/l | NDa
| 8.4 | |
CBS ligands | δ-ALA 0.5 mmol/lb
| Not measured | |
L-serine 100 mmol/l | NDa
| NDa
| |
SAM 1 mmol/l | 0.05 | 0.05 |
Effect of chaperones on structure and function of CBS mutants
topology of the mutants | mutants | δ-ALA | betaine | glycerol | taurine | ||||
---|---|---|---|---|---|---|---|---|---|
0 mM | 0.5 mM | 0 mM | 100 mM | 0 mM | 750 mM | 0 mM | 50 mM | ||
buried mutants | p.G305R | 213.4a
| 277.8a
| 137.2a
| 187.2a
| 276.6 | 264.8 | 199.8 | 237.8 |
p.G307S | 250.2a
| 311.9a
| 230.2 | 226.4 | 102.5a
| 143.1a
| 258.7 | 253.3 | |
p.P422L | 86.0a
| 100.5a
| 28.2 | 27.2 | 23.5a
| 47.3a
| 19.0 | 20.0 | |
p.I278T | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.E144K | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.G148R | N.D. | N.D. | N.D. | N.D. | 1.9a
| 20.8a
| N.D. | N.D. | |
p.R266K | 28.7a
| 65.8a
| 15.0a
| 19.3a
| 11.8a
| 28.6a
| 11.1 | 8.4 | |
p.T262R | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.N228K | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.L539S | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.W409_G453del | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.C165Y | 152.3a
| 270.2a
| 112.4a
| 155.7a
| 125.3a
| 172.3a
| 100.5 | 109.9 | |
p.S466L | 133.0a
| 199.0a
| 47.1 | 55.0 | 53.4a
| 74.9a
| 88.7 | 81.8 | |
solvent accessible mutants | p.H65R | N.D. | N.D. | N.D. | N.D. | 4.6a
| 18.8a
| N.D. | N.D. |
p.R369C | 9.7a
| 47.7a
| 17.7a
| 19.7a
| 10.9a
| 19.4a
| 19.1 | 17.8 | |
p.T191M | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.A114V | 127.3a
| 207.5a
| 69.6 | 59.2 | 113.4 | 130.2 | 127.0 | 115.9 | |
p.P78R | 45.9a
| 72.6a
| 7.2a
| 13.1a
| 9.7a
| 34.0a
| 4.2 | 4.2 | |
p.R125Q | 7.3a
| 64.2a
| 6.4a
| 13.6a
| 15.1a
| 22.9a
| 6.5 | 7.8 | |
p.K102N | 24.2a
| 45.4a
| 6.5a
| 9.3a
| 8.4a
| 22.7a
| 12.6 | 15.6 | |
p.E176K | 5.8 | 2.2 | 13.7 | 14.2 | 12.6a
| 29.2a
| 7.3 | 6.8 | |
p.V180A | 41.9a
| 76.6a
| 11.6a
| 16.2a
| 12.5a
| 30.9a
| 20.6 | 19.9 | |
p.I435T | 72.3 | 50.9 | 76.4a
| 109.8a
| 125.8a
| 186.8a
| 109.1 | 96.7 | |
p.P49L | 224.3a
| 256.0a
| 160.0a
| 198.2a
| 88.2a
| 109.3a
| 156.1 | 163.1 | |
p.E302K | 175.8a
| 233.8a
| 247.5 | 213.2 | 234.1 | 218.0 | 187.4 | 182.8 | |
p.R439Q | 148.2 | 153.5 | 219.1 | 247.3 | 329.2 | 233.1 | 189.4 | 204.8 | |
p.D444N | 180.1 | 208.4 | 261.0 | 297.8 | 377.6 | 350.8 | 291.0 | 300.5 | |
wild-type CBS | 100.0a
| 110.2a
| 100.0a
| 109.3a
| 100.0a
| 120.6a
| 100.0a
| 105.4a
|
topology of the mutants | mutants | δ-ALA | betaine | glycerol | taurine | ||||
---|---|---|---|---|---|---|---|---|---|
0 mM | 0.5 mM | 0 mM | 100 mM | 0 mM | 750 mM | 0 mM | 50 mM | ||
buried mutants | p.G305R | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. |
p.G307S | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.P422L | 43.4a
| 65.0a
| 48.4 | 63.2 | 61.4a
| 150.0a
| 61.7 | 64.0 | |
p.I278T | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.E144K | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.G148R | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.R266K | 19.5a
| 66.1a
| 21.7a
| 33.8a
| 17.1a
| 76.1a
| 48.7 | 51.0 | |
p.T262R | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.N228K | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.L539S | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.W409_G453del | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.C165Y | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.S466L | 260.5a
| 382.5a
| 280.5 | 360.0 | 229.5a
| 453.0a
| 226.0 | 173.6 | |
solvent accessible mutants | p.H65R | N.D. | N.D. | N.D. | N.D. | 2.5a
| 15a
| N.D. | N.D. |
p.R369C | 2.1a
| 6.1a
| 1.8a
| 2.7a
| 1.7a
| 6.2a
| 2.0 | 1.9 | |
p.T191M | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |
p.A114V | 69.5a
| 101.7a
| 69.8 | 71.0 | 75.8 | 100.8 | 58.8 | 60.3 | |
p.P78R | 9.4a
| 24.0a
| 14.1a
| 19.9a
| 7.8a
| 40.7a
| 2.8 | 1.6 | |
p.R125Q | 2.0a
| 8.1a
| 2.6a
| 3.3a
| 1.5a
| 4.1a
| 6.8 | 2.7 | |
p.K102N | 8.4a
| 11.7a
| 9.9a
| 12.7a
| 5.5a
| 21.9a
| 9.2 | 6.8 | |
p.E176K | 8.6 | 8.5 | 1.5 | 1.5 | 2.7a
| 17.5a
| 7.3 | 6.8 | |
p.V180A | 12.4a
| 35.2a
| 9.0a
| 12.0a
| 13.0a
| 55.7a
| 25.8 | 26.8 | |
p.I435T | 132.1 | 135.7 | 174.6a
| 191.0a
| 215.3a
| 340.3a
| 205.8 | 177.3 | |
p.P49L | 119.5a
| 172.5a
| 158.5a
| 214.5a
| 119.0a
| 158.5a
| 92.8 | 94.0 | |
p.E302K | 107.0a
| 152.0a
| 78.4 | 82.6 | 140.7 | 137.7 | 134.9 | 153.0 | |
p.R439Q | 138.7 | 150.6 | 139.9 | 180.4 | 131.9 | 152.6 | 94.6 | 69.1 | |
p.D444N | 220.8 | 183.5 | 196.1 | 223.9 | 179.4 | 205.0 | 124.8 | 137.5 | |
wild type CBS | 107.4a
| 118.0a
| 135.1a
| 150.2a
| 129.3a
| 153.2a
| 111.7a
| 109.0a
|