Table 1.
Effects of Substitutions on Free CheA Autokinase Activitya
| kinase activity | kinase activity | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| position | domain | 2° struct. | Cys | Cys-F1 | labeling efficiency | position | domain | 2° struct. | Cys | Cys-F1 | labeling efficiency |
| I5C | P1 | α1 | (+) | (+) | 0.88 | I388C | P4 | α11 | (+) | [-] | 0.88 |
| Q10C | P1 | α1 | [++] | (+) | 0.86 | H401C | P4 | α11 | (+) | (+) | 0.80 |
| D17C | P1 | α1 | (+) | (+) | 0.91 | A412C | P4 | α12 | (+) | (+) | 0.81 |
| Q25C | P1 | α1 | (+) | (+) | 0.80 | E425C | P4 | β8 | (+) | [++] | 0.89 |
| A37C | P1 | α2 | (+) | (+) | 0.97 | T436C | P4 | β9 | (+) | (+) | 0.87 |
| A42C | P1 | α2 | [-] | [-] | 0.88 | R446C | P4 | α13 | (+) | [-] | 0.86 |
| T60C | P1 | L | (+) | (+) | 1.02 | Q454C | P4 | L | (+) | (+) | 0.94 |
| E75C | P1 | α3 | (+) | (+) | 0.94 | E460C | P4 | L | (+) | (+) | 0.87 |
| Q82C | P1 | L | (+) | (+) | 1.03 | D465C | P4 | α14 | (+) | (+) | 0.80 |
| E93C | P1 | α4 | (+) | (+) | 1.10 | M469C | P4 | L | (+) | (+) | 0.97 |
| E109C | P1 | L | (+) | (+) | 1.11 | E480C | P4 | L | (+) | [-] | 0.99 |
| A114C | P1 | α5 | (+) | (+) | 0.85 | V485C | P4 | L | [-] | [-] | 1.01 |
| Q125C | P1 | α5 | (+) | (+) | 0.81 | R497C | P4 | α16 | [++] | (+) | 0.94 |
| V168C | P2 | β1 | (+) | (+) | 1.04 | S514C | P4 | L | (+) | (+) | 0.86 |
| R171C | P2 | L | (+) | (+) | 0.96 | L521C | P4 | β11 | (+) | (+) | 0.82 |
| V177C | P2 | α6 | (+) | (+) | 0.95 | E539C | P5 | β13 | (+) | (+) | 0.84 |
| E182C | P2 | α6 | (+) | (+) | 0.89 | L545C | P5 | α17 | (+) | (+) | 0.99 |
| T189C | P2 | L | (+) | (+) | 0.92 | E550C | P5 | β14 | (+) | (+) | 0.88 |
| D192C | P2 | L | (+) | (+) | 0.86 | E557C | P5 | L | (+) | (+) | 0.82 |
| V216C | P2 | α7 | (+) | (+) | 0.87 | L562C | P5 | β15 | (+) | (+) | 0.86 |
| V220C | P2 | α7 | (+) | (+) | 0.84 | G565C | P5 | L | (+) | (+) | 0.80 |
| A223C | P2 | L | (+) | (+) | 0.95 | E570C | P5 | β16 | (+) | (+) | 0.90 |
| K230C | P2 | β3 | (+) | (+) | 1.01 | V579C | P5 | β17 | (+) | (+) | 0.85 |
| T279C | P3 | L | (+) | [-] | 0.88 | D588C | P5 | L | (+) | (+) | 1.04 |
| I292C | P3 | α8 | (+) | [-] | 1.03 | T592C | P5 | L | (+) | (+) | 0.96 |
| S303C | P3 | α8 | (+) | (+) | 0.86 | Q596C | P5 | L | (+) | (+) | 0.93 |
| E311C | P3 | L | (+) | (+) | 0.81 | I598C | P5 | β18 | (+) | (+) | 0.95 |
| G318C | P3 | α9 | (+) | (+) | 0.90 | R607C | P5 | β19 | (+) | (+) | 0.80 |
| G325C | P3 | α9 | (+) | (+) | 0.87 | I617C | P5 | β19 | (+) | (+) | 1.03 |
| R332C | P3 | α9 | (+) | (+) | 0.84 | Q621C | P5 | β19 | (+) | (+) | 0.92 |
| S340C | P3 | α9 | (+) | (+) | 0.81 | L627C | P5 | L | (+) | (+) | 0.98 |
| S351C | P4 | α10 | (+) | [-] | 0.68 | S629C | P5 | α19 | (+) | (+) | 0.84 |
| D359C | P4 | α10 | (+) | [-] | 1.00 | G636C | P5 | L | (+) | (+) | 0.93 |
| G365C | P4 | L | [-] | [-] | 0.88 | G644C | P5 | L | (+) | (+) | 0.89 |
| E369C | P4 | β6 | (+) | (+) | 0.84 | S647C | P5 | L | (+) | (+) | 0.89 |
a
The autophosphorylation activity of each single Cys CheA was tested in vitro without (Cys) and with (Cys-F1) the attached 5-FM probe. The resulting kinase activities were operationally defined as (+), [-], or [++] corresponding to normal, inhibited (less than 25% normal), or super-activated (over 250% normal) relative to Cysless CheA, respectively. Labeling efficiency is the final extent of 5FM labeling and has an estimated error of ± 10%.