Illumination of the Structure and Function of PptA, a Novel Type of Polyphosphate Granule Attached Protein in Organelle-like Polyphosphatosomes of Ralstonia eutropha H16

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In this project we will determine the structure and function of PptA, a protein of R. eutropha that is characterized by an ubiquitously occurring (in all kingdoms of life) CHAD (conserved histidine alpha-helical) domain at the edge of P- and N-metabolism. PptA has two remarkable proterties: (i) it´s CHAD domain confers a specific binding to polyP granules in a species independent manor and (ii) it interacts with the key enzyme of N-metabolism, glutamine synthetase GlnA1. Here, we will determine whether arginines of conserved sequence motifs (isoelectric point of PptA is 11.5!) or other residues of PptA, that are conserved in CHAD proteins, are responsible for interaction with the polyanion polyP and/or GlnA1 (IEP 5.4). The strength and stoichiometry of the PptA-GlnA1, PptA-polyP and GlnA1-polyP interaction will be determined by isothermic colometric titration (ITC) and/or surface plasmon resonance (SPR). The influence of the PptA-GlnA1 interaction on the oligomeric state and activity and of GlnA1 in the presence of different effector molecules will be determined by light scattering technique.