Table 1.
Effects of Arginine and Cysteine Substitutions on Receptor Kinase and Methylation Activitiesa
| A. Arginine Substitutions | ||||
|---|---|---|---|---|
| kinase activity |
methylation activity |
|||
| position | (–)Asp | (+)Asp | (–)Asp | (+)Asp |
| R188 (WT) | 1.00 ± 0.03 | 0.00 ± 0.00 | 1.00 ± 0.13 | 2.63 ± 0.08 |
| F189R | 2.05 ± 0.45 | 0.05 ± 0.03 | 0.29 ± 0.05 | 0.40 ± 0.05 |
| A190R | 0.77 ± 0.12 | 0.07 ± 0.03 | 0.09 ± 0.02 | 0.03 ± 0.00 |
| Q191R | 0.59 ± 0.18 | 0.04 ± 0.03 | 0.32 ± 0.02 | 0.92 ± 0.09 |
| W192R | 3.55 ± 0.77 | 0.00 ± 0.01 | 0.28 ± 0.04 | 0.58 ± 0.04 |
| Q193R | 0.00 ± 0.02 | 0.03 ± 0.04 | 0.01 ± 0.06 | 0.08 ± 0.04 |
| L194R | 0.03 ± 0.02 | 0.00 ± 0.01 | 0.20 ± 0.04 | 0.31 ± 0.06 |
| V208R | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.09 ± 0.07 | 0.10 ± 0.03 |
| W209R | 0.03 ± 0.05 | 0.03 ± 0.03 | 2.37 ± 0.22 | 2.57 ± 0.20 |
| F210R | b | b | b | b |
| G211R | 0.34 ± 0.05 | 0.01 ± 0.20 | 0.61 ± 0.10 | 0.95 ± 0.03 |
| I212R | b | b | b | b |
| R213 (WT) | 1.00 ± 0.03 | 0.00 ± 0.00 | 1.00 ± 0.13 | 2.63 ± 0.08 |
| H214R | 0.63 ± 0.24 | 0.04 ± 0.02 | 0.24 ± 0.06 | 0.05 ± 0.03 |
| B. Cysteine Subsitutions | ||||
|---|---|---|---|---|
| kinase activity |
methylation activity |
|||
| position | (–)Asp | (+)Asp | (–)Asp | (+)Asp |
| R188 (WT) | 1.00 ± 0.03 | 0.00 ± 0.00 | 1.00 ± 0.13 | 2.63 ± 0.08 |
| R188C | 0.38 ± 0.03 | 0.05 ± 0.01 | 0.92 ± 0.13 | 1.80 ± 0.16 |
| F189C | 3.90 ± 0.46 | 0.06 ± 0.02 | 0.43 ± 0.01 | 1.42 ± 0.15 |
| A190C | 0.22 ± 0.02 | 0.03 ± 0.00 | 0.34 ± 0.03 | 0.77 ± 0.03 |
| Q191C | 0.46 ± 0.08 | 0.03 ± 0.02 | 0.58 ± 0.09 | 1.18 ± 0.04 |
| W192C | 0.44 ± 0.16 | 0.02 ± 0.02 | 0.68 ± 0.06 | 1.22 ± 0.17 |
| Q193C | 0.02 ± 0.01 | 0.01 ± 0.01 | 1.09 ± 0.15 | 1.39 ± 0.08 |
| L194C | 0.01 ± 0.01 | 0.02 ± 0.02 | 0.14 ± 0.02 | 0.01 ± 0.11 |
| V208C | 1.74 ± 0.39 | 0.05 ± 0.03 | 0.40 ± 0.02 | 1.35 ± 0.19 |
| W209C | 0.03 ± 0.02 | 0.04 ± 0.02 | 0.24 ± 0.05 | 0.37 ± 0.10 |
| F210C | 1.22 ± 0.37 | 0.02 ± 0.02 | 0.25 ± 0.07 | 0.97 ± 0.12 |
| G211C | 0.45 ± 0.09 | 0.01 ± 0.01 | 0.12 ± 0.01 | 0.33 ± 0.03 |
| I212C | 0.07 ± 0.03 | 0.04 ± 0.16 | 0.53 ± 0.18 | 1.05 ± 0.07 |
| R213C | 0.34 ± 0.01 | 0.00 ± 0.00 | 0.19 ± 0.04 | 0.62 ± 0.12 |
| H214C | 0.23 ± 0.04 | 0.03 ± 0.03 | 0.26 ± 0.01 | 1.06 ± 0.09 |
Shown are the activities of (A) arginine-substituted and (B) cysteine-substituted receptors in two antisymmetric functional assays, which together identify receptors in the on- or off-state. In both assays, activities are measured in triplicate and expressed relative to the wild-type apo receptor. The kinase assay reconstitutes receptor-containing membranes with the purified, soluble pathway components CheA, CheW, and CheY to form the receptor-kinase signaling complex. Following addition of γ-[32P]-ATP to trigger the kinase reaction, the initial rate of [32P]-phospho-CheY formation at 25 °C is measured under conditions where the autophosphorylation of CheA is rate-determining, both in the absence and presence of the attractant l-aspartate (Asp) (see Materials and Methods and ref 27). The methylation assay reconstitutes receptor-containing membranes with the adaptation enzyme CheR. Following the addition of [3H]-S-adenosyl methionine, the initial rate of receptor methyl esterification is measured at 30 °C (see text and ref 25). The receptor on-state is recognized by its activation of the cytoplasmic kinase CheA and inhibition of the cytoplasmic methylation enzyme CheR, while the off-state is recognized by its inhibition of kinase activity and stimulation of methylation activity.
Receptors that failed to accumulate in cell membranes, presumably because membrane insertion or protein stability is disrupted.