Table 1.
Disulphide cross-linking between CheW and Tsr.
| CheW
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| S15 | I33 | V36 | E38 | I39 | G41 | R62 | V87 | V108 | ||
| Tsr | S366 | − | − | − | − | − | − | − | − | − |
| N376* | − | − | − | − | − | − | − | − | − | |
| V384 | + | − | − | − | − | − | − | − | − | |
| R388 | − | + | − | − | − | − | − | − | − | |
| A389 | − | − | − | − | − | − | − | − | − | |
| E391 | ++ | − | − | − | − | + | − | − | − | |
| V398** | + | − | − | − | − | − | ++ | − | − | |
| R409 | − | − | − | − | − | − | − | − | − | |
| S410* | − | − | − | − | − | − | − | − | − | |
Assays were done with the equivalent Tar replacements.
Assays were done with either Tsr or Tar replacement.
UU1581 cells expressing cysteine-substituted Tsr or Tar at physiological levels (0.45 μM sodium salicylate, pCS12 plasmid derivatives) and cysteine-substituted CheW at over-expression levels (50–100 μM IPTG, pPA770 plasmid derivatives) were treated with diamide, and the crosslinking products were analysed by immunoblotting with an anti-CheW polyclonal antibody. Band intensity was determined with the software ImageQuant (Amersham). The occurrence of covalent binding between Tsr and CheW under the assayed conditions was calculated as the percentage of crosslinking product relative to the total amount of Tsr in each sample. Categories correspond to − (<20%), + (20–40%) or ++ (>40%).