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Supplementary figures

Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA-DrrA from Thermotoga maritima, by J. E. Foster, Q. Sheng, J. R. McClain, M. Bures, T. I. Nicas, K. Henry, M. E. Winkler and R. Gilmour

Microbiology vol. 150, part 4, pp. 885 - 896

Supplementary figures S1–S4. See below for legends.

Fig. S1. Purification of His-HpkA77 and His-DrrA expressed in E. coli. Proteins were expressed, purified by nickel-Sepharose chromatography, and analysed by 4–20 % SDS-PAGE. Gels were stained with Coomassie blue. Lane 1, purified His-HpkA77; lane 2, wide-range protein molecular mass standards (kDa); lane 3, purified His-DrrA; lane 4, multicolour protein molecular mass standards (kDa).

Fig. S2. Autophosphorylation of His-HpkA77. Initial velocities of autophosphorylation were determined as described in Methods. The data are means from three independent experiments. The fit was obtained using a K_m of 20 µM and a V_max of 0.044 nM min^–1 (see Table 1 in main paper).

Fig. S3. Effect of increasing His-HpkA77 concentrations on DrrA-P product formation in the HpkA–DrrA combined assay. The combined assay was performed as described in Methods. Reactions contained His-HpkA77 concentrations varying from 20 to 80 nM in the presence of 4 µM DrrA and 200 µM ATP. Data were analysed by linear regression.

Fig. S4. [³²P]DrrA formation as a function of ATP or DrrA concentration in the HpkA–DrrA combined assay. (a) Initial velocity versus ATP concentration. Rates were determined in reactions containing 20 nM His-HpkA77 and 4 µM His-DrrA and analysed by SDS-PAGE (see Methods). (b) Initial velocity versus His-DrrA concentration. Rates were determined in reactions containing 20 nM His-HpkA77 and 80 µM ATP and analysed by the filter format (see Methods). The data are means of at least three independent experiments. Curves were fitted to the Michaelis–Menten equation; apparent K_m and V_max values are listed in Table 1 in the main paper.

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