| Alginate is an important virulence factor which produced by human opportunistic pathogen Pseudomonas aeruginosa and it is also one of major polysaccharide which Pseudomonas aeruginosa can secreted. Alginate can be obtained from mucoid type Pseudomonas aeruginosa witch can lead to high morbidity and mortality of cystic fibrosis lung diease. This polysaccharide can help Pseudomonas aeruginosa survive from serious environment in cystic fibrosis by constitute matix. At the same time, drug resistance of Pseudomonas aeruginosa has has great relationship with alginate. Research shows that this polysaccharide can get rid of neutrophils and radical which can be used to kill pathogen.Due to this polysaccharide is closely related to the pathogenicity of P. aeruginosa, so the study on the synthesis of this polysaccharide are more detailed Current research shows that there are 13 kinds of proteins participated in the synthesis of alginate. They are from a complex which stretch over the intracellular membranes, periplasmic space and outer membrane to function. Glycosyltransferase Alg8 and c-di-gmp binding protein Alg44 are essential to the synthesis of alginate. When knockout Alg8 and Alg44, the bacterial can’t secrete alginate. From that we can see Alg44 plays an important role in alginate synthesis.But the specific regulatory mechanism is not clear. So we chose Alg44 as our target protein. We hope to elucidate the function mechanism of this protein through the protein structure. Alg44 is a single transmembrane protein, which N-terminal is located in the cytoplasm and it is a pilz domain, a typical c-di-gmp binding domain. Research shows that the binding of c-di-gmp plays a key role in alginate synthesis. C-terminal is located in periplasm, it may play a role in forming the large protein complex. We try to use molecular cloning and membrane protein purification method to get full-length of our target protein, but unfortunately, we failed. We also try to get the protein of different domains including N-terminal and C-terminal domain. We obtained soluble pilz domain from 16 to 122, and fortunately we obtained the complex of no his-tag protein and c-di-gmp. In Shanghai Synchrotron Radiation Facility, we gathered the crystal diffraction data and resolved the structure of alg44 pilz domain with CCP4, coot, phenix and CNS.In the typical pilz domain, one monomer usually binds two c-di-gmp. But to our surprise, in our structure we saw that a monomer bind three c-di-gmp, one of the c-di-gmp didn’t interact with protein. We inferred that maybe this phenomenon was caused by the excessive amount of c-di-gmp. So we changed the proportion between protein and c-di-gmp from 1:5 to 1:3. The binding ratio of protein monomer and c-di-gmp became to 1:2. Two molecular c-di-gmp paralleled with protein. We first observed the polymorphism of c-di-gmp and we thought this was not accidental. C-di-gmp can help the folding of protein through different ways and it hasn’t been reported before.In the crystal structure of pilz domain, one asymmetric unit had two monomer, so it is a dimer.Through the analytical column, we find that, the protein is also a dimer in solution. In order to clarify whether the formation of the dimer is due to the c-di-gmp, we designed c-di-gmp binding site mutants according to the structure, including Q18A, R17A and Q18AR17A. By the analysis of mutants, we found that c-di-gmp didn’t influence the formation of dimer.C-terminal domain in periplasm was insoluble. We tried to optimize the culture condition and change the medium, but they were not helpful. We also tried to use protein-protein interaction to get soluble protein, but we still failed. In the end we used the method of purification of inclusion body, we obtained the soluble C-terminal domain of the protein. |
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