Browsing by Subject "MCP"

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Interaktion des Photosensors Ppr aus Rhodocista centenaria mit Proteinkomponenten der chemotaktischen Signaltransduktion
(2008) Kreutel, Sven; Kuhn, Andreas
Rhodocista centenaria (previously known as Rhodospirillum centenum) is a photosynthetic alpha-proteobacterium which exhibits a unique phototactic response in respect of the direction of light. In this work, the focus is on the potential photoreceptor Ppr and its C-terminal histidine kinase Pph to identify putative binding partners in the signal transduction pathway. The results of overexpression experiments with the Ppr-receptor or the Pph-domain in E. coli indicated that there may be an interaction between the photosensor and the chemotactic signalling pathway. Even cells expressing only small amounts of the R. centenaria proteins showed no chemotactic response at all, whereas uninduced cells exhibited normal chemotactic response on swarm plates as well as in capillary assays. To investigate whether the receptor interacts with components of the chemotactic pathway, the Ppr-protein and the Pph-domain as well as the chemotactic proteins CheW and CheAY of R. centenaria were heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. Binding experiments were carried out by using an IAsys biosensor cuvette system. The results indicated that the kinase domain Pph binds to the chemotactic linker protein CheW with a dissociation constant of 0.13 ± 0.026 μM. Pull-down experiments were made to verify this finding and to investigate the role of ATP in the binding process. The results confirmed our previous observations but in contrast to the complex formation in the E. coli chemotactic pathway, the binding of the C-terminal histidine kinase to CheW was ATP-dependent. To study whether the kinase domain also binds CheW in vivo, expression and coelution experiments with tagged Pph-protein were carried out in R. centenaria. The findings suggest that the complex formation occurs in vivo as well as in vitro. From the data of the E. coli chemotactic complex formation it is well known that CheA is part of the trimeric complex which consists of MCP-receptors, CheW and CheA. To analyse this, pulldown experiments with all three proteins (Pph, CheW and CheAY) were performed. The results clearly showed a participation of CheAY in the formation of the complex with CheW and the kinase Pph. It is known that the photoreceptor Ppr is autophosphorylated during its light induced photocycle. We therefore examined whether the kinase domain is sufficient for this autophosphorylation reaction and whether CheAY could function as a phosphate acceptor. Our results confirmed the hypothesis that the kinase domain is sufficient for autophosphorylation and that the Pph-protein assists CheAY to take over phosphate groups. Taken together, the results in combination with data from the literature lead to a detailed working model for the function of the photoreceptor Ppr and the signal transduction pathway.