Mutation of the conserved Asp residue affects the inhibitory activity of VieB. GST-VieS-C was incubated with 32P-ATP-γ in the presence of equimolar VieA-His6 (lane 1) and either 5 μM wild-type VieB (lane 2), VieB D62A (lane 3), or VieB D62E (lane 4) for 30 minutes at 30°C. Samples were stopped with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. Proteins labeled with 32P were observed by radioautography. Figure panels are from the same experiment and exposed for the same amount of time. The radioautographs shown are a representative of four replicates. Standard deviation is shown below. Stars denotes a significant P-value < 0.05 determine by a Mann–Whitney U test.