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

在这个项目中，我们将确定PPTA的结构和功能，PPTA是一种富营养化罗氏杆菌的蛋白质，其特征是在P-和N-代谢的边缘普遍存在(在所有生命王国中)查德(保守的组氨酸α-螺旋)结构域。PPTA有两个显著的特性：(I)它的S乍得结构域与物种无关的庄园中的息肉颗粒具有特异性结合；(Ii)它与N代谢的关键酶-谷氨酰胺合成酶GlnA1相互作用。在这里，我们将确定保守序列基序的精氨酸(PPTA的等电点为11.5！)或在乍得蛋白中保守的PPTA的其他残基，负责与聚阴离子息肉和/或GlnA1的相互作用(IEP 5.4)。PPTA-GlnA1、PPTA-POLIP和GlnA1-POLIP相互作用的强度和化学计量比将通过等温比色滴定(ITC)和/或表面等离子体共振(SPR)来确定。PPTA-GlnA1相互作用对低聚态和活性的影响以及不同效应分子存在时GlnA1的影响将通过光散射技术来确定。

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.