Table 4.
Effect of Engineered Cysteines and Disulfides on in Vitro Methylation
| relative methylation ratec | ||||||
|---|---|---|---|---|---|---|
| receptor | conditiona | fraction S– Sb | (−) aspartate (/WT (−)Asp) |
(+) aspartate (/WT (−)Asp) |
maximum activityd (/WT (+) Asp) |
aspartate regulatione (/WT) |
| WT | (−) | na | 1.0 ±0.1 | 2.3 ± 0.2 | 1.0 | 1.0 |
| red | na | 1.0 ±0.1 | 2.3 ±0.2 | 1.0 | 1.0 | |
| ox (O2, I2) | na | 1.0 ±0.1 | 2.3 ± 0.2 | 1.0 | 1.0 | |
| M75C | red | 3.2 ± 0.5 | 5.1 ± 1.2 | 2.2 | 0.4 | |
| ox (O2) | 0.9 ±0.1 | 2.4 ± 0.2 | 4.2 ± 0.4 | 1.8 | 0.6 | |
| A72C | red | 2.0 ± 0.4 | 3.4 ± 0.4 | 1.5 | 0.5 | |
| ox (I2) | 0.8 ±0.1 | 1.7 ±0.5 | 3.6 ± 1.1 | 1.6 | 0.9 | |
| S68C | red | 2.2 ± 0.8 | 5.5 ±0.9 | 2.3 | 1.1 | |
| ox (I2) | 0.9 ±0.1 | 2.4 ± 0.8 | 2.8 ±0.5 | 1.2 | 0.1 | |
| I65C | red | 1.6 ±0.3 | 3.4 ± 1.1 | 1.4 | 0.8 | |
| ox (O2) | 0.8 ±0.1 | 1.1 ±0.5 | 3.3 ±0.6 | 1.4 | 1.5 | |
| L61C | red | 2.0 ± 0.6 | 3.5 ± 1.1 | 1.4 | 0.4 | |
| ox (O2) | 0.6 ±0.1 | 1.7 ± 1.0 | 2.6 ±0.6 | 1.1 | 0.4 | |
| N36C | red | 4.1 ± 1.1 | 9.9 ±2.1 | 4.3 | 1.1 | |
| ox (O2) | 0.9 ±0.1 | 5.3 ± 1.5 | 11 ±2.2 | 4.6 | 0.8 | |
| L33C | red | 4.2 ± 0.5 | 7.7 ± 0.6 | 3.3 | 0.6 | |
| ox (O2) | 1.0 ±0.1 | 2.5 ± 0.5 | 3.6 ± 0.8 | 1.6 | 0.3 | |
| L29C | red | 3.7 ± 0.4 | 7.1 ±0.5 | 3.1 | 0.7 | |
| ox (O2) | 0.9 ±0.1 | 3.5 ±0.7 | 7.7 ± 1.1 | 3.3 | 0.9 | |
| Q22C | red | 12 ± 1.8 | 18 ±4.5 | 7.9 | 0.4 | |
| ox (I2) | 0.9 ±0.1 | 13 ± 1.4 | 7.4 ± 1.0 | 3.2 | 0.0 | |
| F18C | red | 4.5 ±0.6 | 6.4 ± 0.2 | 2.8 | 0.3 | |
| ox (I2) | 0.9 ±0.1 | 1.6 ±0.3 | 4.2± 1.3 | 1.8 | 1.3 | |
| L11C | red | 3.7 ± 0.4 | 7.0 ± 0.7 | 3.0 | 0.7 | |
| ox (I2) | 0.7 ±0.1 | 1.0 ±0.1 | 0.6 ± 0.2 | 0.3 | 0.0 | |
| V8C | red | 0.4 ± 0.04 | 0.9 ± 0.2 | 0.4 | 0.9 | |
| ox (O2) | 0.8 ±0.1 | 0.2 ±0.1 | 0.4 ± 0.2 | 0.2 | 0.5 | |
| V7C | red | 2.1 ±0.1 | 3.3 ±0.1 | 1.4 | 0.5 | |
| ox (O2) | 0.6 ±0.1 | 0.1 ±0.1 | 0.2 ±0.1 | 0.1 | 0.9 | |
| R4C | red | 1.1 ±0.2 | 1.6 ±0.1 | 0.7 | 0.4 | |
| ox (O2) | 0.7 ±0.1 | 0.3 ±0.3 | 0.3 ± 0.2 | 0.1 | 0.1 | |
| no Tar | (–) | na | 0 | 0 | 0 | 0 |
Isolated membranes were treated as described in Materials and Methods: no treatment (−); reduction by dithiothreitol (red); oxidation (ox) by molecular iodine (I2) or oxygen (O2).
Fraction of receptor population linked by disulfide bonds after oxidation.
Rate of in vitro receptor methylation by the methyltransferase CheR in the absence or presence of ligand, relative to the rate observed for the unliganded wild-type receptor subjected to the same chemical treatment (1.8 pmol of methyl groups min−1). All rates are normalized to the same receptor concentration (3 µM dimer).
Rate of in vitro receptor methylation by CheR in the presence of aspartate, relative to rate observed for the aspartate-occupied wild-type receptor subjected to the same chemical treatment (4.2 pmol of methyl groups min−1 for 3 µM receptor dimer).
Effect of aspartate on the rate of in vitro receptor methylation by CheR, relative to the effect of aspartate on the wild-type receptor subjected to the same chemical treatment (2.3-fold up-regulation upon aspartate addition). Calculated as the ratio {([rate (+) Asp]/[rate (−) Asp]) −1}/1.3, yielding a range between 0 and 1 for no aspartate regulation or wild-type regulation, respectively.