Table 3.
Effect of Engineered Cysteines and Disulfides on in Vitro Phosphorylation
| relative phosphorylation ratec | ||||||
|---|---|---|---|---|---|---|
| receptor | conditiona | fraction S–Sb | (−) aspartate (/WT (+)Asp) |
(+) aspartate (/WT (+)Asp) |
maximum activityd (/WT (−) Asp) |
aspartate regulatione (/WT) |
| WT | (−) | na | 50 ±5 | 1.0 ±0.2 | 1.0 | 1.0 |
| red | na | 50 ± 5 | 1.0 ±0.2 | 1.0 | 1.0 | |
| ox (O2, I2) | na | 50 ± 5 | 1.0 ±0.2 | 1.0 | 1.0 | |
| Me | na | 50 ± 5 | 1.0 ±0.2 | 1.0 | 1.0 | |
| M75C | red | 38 ±2 | 0.6 ±0.1 | 0.8 | 1.3 | |
| ox (O2) | 0.9 ± 0.1 | 25 ±4 | 0.8 ±0.1 | 0.5 | 0.7 | |
| A72C | red | 61 ± 1 | 1.0 ±0.5 | 1.2 | 1.2 | |
| ox (I2) | 0.8 ± 0.1 | 82 ± 2 | 2.4 ±0.1 | 1.6 | 0.7 | |
| S68C | red | 70 ± 4 | 0.6 ± 0.2 | 1.4 | 2.3 | |
| ox (I2) | 0.9 ± 0.1 | 11 ±2 | 0.5 ±0.1 | 0.2 | 0.4 | |
| I65C | red | 39 ± 5 | 0.8 ±0.5 | 0.8 | 1.0 | |
| ox (O2) | 0.8 ± 0.1 | 12 ± 2 | 1.3 ±0.7 | 0.2 | 0.2 | |
| L61C | red | 34 ± 2 | 0.6 ±0.5 | 0.7 | 1.1 | |
| ox (O2) | 0.6 ± 0.1 | 19 ± 1 | 0.7 ± 0.4 | 0.4 | 0.6 | |
| N36C | red | 76 ± 12 | 1.3 ±0.2 | 1.5 | 1.2 | |
| ox (O2) | 0.9 ± 0.1 | 43 ± 2 | 1.3 ±0.1 | 0.9 | 0.7 | |
| L33C | red | 3 ± 1 | 0.7 ± 0.3 | 0.1 | 0.1 | |
| ox (O2) | 1.0 ± 0.1 | 16 ± 1 | 2.8 ± 0.7 | 0.3 | 0.1 | |
| L29C | red | 37 ± 3 | 0.8 ±0.1 | 0.7 | 0.9 | |
| ox (O2) | 0.9 ± 0.1 | 37 ± 9 | 0.9 ± 0.2 | 0.7 | 0.8 | |
| Q22C | red | 1 ± 0.5 | 0.3 ±0.1 | 0.02 | 0.04 | |
| ox (I2) | 0.9 ± 0.1 | 1 ± 0.2 | 0.2 ±0.1 | 0.02 | 0.04 | |
| red (Me) | 28 ± 3 | 2.8 ±0.3 | 0.6 | 0.2 | ||
| ox (Me,I2) | 1.0 ± 0.1 | 7 ± 1 | 1.0 ±0.1 | 0.1 | 0.1 | |
| F18C | red | 1 ± 0.2 | 0.3 ± 0.2 | 0.02 | 0.04 | |
| ox (I2) | 0.9 ± 0.1 | 19 ± 1 | 0.7 ± 0.3 | 0.4 | 0.5 | |
| L11C | red | 137 ±5 | 1.9 ±0.1 | 2.7 | 1.4 | |
| ox (I2) | 0.7 ± 0.1 | 2 ± 0.4 | 1.0 ±0.3 | 0.04 | 0.03 | |
| V8C | red | 65 ± 7 | 1.1 ±0.2 | 1.3 | 1.2 | |
| ox (O2) | 0.8 ± 0.1 | 5 ± 2 | 0.9 ±0.1 | 0.1 | 0.1 | |
| V7C | red | 8± 2 | 0.3 ±0.1 | 0.2 | 0.5 | |
| ox (O2) | 0.6 ± 0.1 | 1 ± 0.1 | 0.4 ±0.1 | 0.02 | 0.01 | |
| R4C | red | 24 ± 3 | 1.1 ±0.1 | 0.5 | 0.4 | |
| ox (O2) | 0.7 ± 0.1 | 2 ± 0.5 | 1.0 ±0.1 | 0.04 | 0.02 | |
| no Tar | (−) | na | 1.0 ± 0.2 | 1.0 ±0.2 | 0.02 | 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); methylation by CheR prior to reduction or oxidation (Me).
Fraction of receptor population linked by disulfide bonds after oxidation.
Rate of CheY phosphorylation by the reconstituted receptor–CheW–CheA ternary kinase complex in the absence or presence of ligand, relative to the rate observed for the aspartate-occupied wild-type complex subjected to the same chemical treatment (1.2 fmol of Pi-CheY s−1). All rates are normalized to the same receptor concentration (3µM dimer). In the presence of ligand, CheY phosphorylation was dominated by the free kinase, yielding the same rate observed in the absence of the receptor (no Tar).
Rate of CheY phosphorylation by the unliganded receptor–CheW–CheA ternary kinase complex, relative to corresponding rate observed for the unliganded wild-type complex subjected to the same chemical treatment (61 fmol of Pj-CheY s−1 for 3 µM receptor dimer).
Effect of aspartate on the rate of CheY phosphorylation by the receptor–CheW–CheA ternary kinase complex, relative to the effect of aspartate on the wild-type complex subjected to the same chemical treatment (50-fold downregulation upon aspartate addition). Calculated as the ratio {([rate (−) Asp]/[rate (+) Asp]) −1}/49, yielding a range between 0 and 1 for no aspartate regulation or wild-type regulation, respectively.