Figure 3: CckA activity on liposomes is density dependent.

(a) CckA autophosphorylation activity was assayed on liposomes. A fixed amount of 5 μM CckA was pre-incubated with increasing amounts of liposomes to obtain surface densities ranging from ∼80 to 800 CckA molecules per liposome. Autophosphorylation was initiated on addition of [γ−³²P] ATP and allowed to proceed for 3 min. Samples quenched in SDS sample buffer were assayed using the nitrocellulose membrane capture assay, followed by phosphorimaging. The activity of each CckA surface density state is normalized to the highest density condition. Representative data is shown above the graph. These data were fit to a sigmoidal curve described in the Methods section. (b) Assays of the kinase activity levels of CckA PAS domain truncations at maximum surface density (∼1,100 CckA per liposome) and low-surface density (∼100 CckA per liposome) showed that the CckA PAS sensor, particularly PAS-A, is required for CckA activity on liposomes. CckA activity levels were normalized to the WT activity at maximum density. Each CckA construct’s domain architecture is shown as a cartoon accompanying its data. Dark grey shading of a domain indicates that the domain is present, while a dashed, white box indicates that the domain is absent. Error bars in a,b represent the range of two experiments.