Study The Structures And Properties Of Demethylase ALKBH5 And Transcription Regulator NalD

Protein is an important biological molecular in the cell, and it is closely related to a wide range of life activities in organisms. Due to the varies of amino acid composition and spatial structure, proteins have different properties and functions. The study of biological activity based on protein structure is important for further insight into various physiological activities and biological phenomena in organisms. In this thesis, the crystal structures of N6-methyladenosine (m6A) demethylase ALKBH5 in zebrafish as well as NalD protein, the transcription regulator of MexAB-OprM mutildrug efflux pump in Pseudomonas aeruginosa, were successfully resolved through protein crystallography. The biological activities and functions of these two proteins were further studied using a series of biochemical techniques according to their structures. In addition, we reconstructed NalD protein to change its DNA binding ability by protein engineering. Thus the modified protein could reconginize the DNA sequence of TetR protein.ALKBH5 belongs to the AlkB family of iron(Ⅱ)/α-ketoglutarate(α-KG) dependent dioxygenases. It could catalysis m6A demethylation to adenine on single stranded RNA. We successfully obtained the crystals of zebrafish ALKBH5, and resolved its structure. The results of structural alignment with another m6A demethylase FTO showed that Lys100 and Pro 175 in ALKBH5 protein may be involved in m6A demethylation reaction. This conclusion was further confirmed by MALDI-TOF-MS experiment. In addition, our experiments showed that the m6A demethylation pathway of ALKBH5 are different from FTO’s. Two intermediates of N6-hydroxymethyl-adenosine (hm6A) and N6-formyladenosine (fm6A) were observed in the FTO-mediated m6A demethylation. Whereas, such intermediates were not observed in demethylation reactions catalyzed by ALKBH5. This study should facilitate our understanding of the substrate-selectivity of the AlkB family proteins, and provide insights for future investigation into the mechanism of m6A demethylation.The MexAB-OprM multidrug efflux pump is an important drug efflux pump in in Pseudomonas aeruginosa, and significantly contributes to its intrinsic resistance. NalD was reported to be the secondary repressor of MexAB-OprM multidrug efflux pump. However, the regulation mechanism by which NalD is activated remains unclear. To study the properties of NalD protein, we expressed and purified NalD protein, and carried out a series of biochemistry experiments. Through thermal shift assay, EMSA, ITC experiments as well as luminescence measurement in vivo, novobiocin was detected to induce NalD protein’s dissocaition from the promoter DNA of MexAB-OprM multidrug efflux pump, and up-regulate the expression of MexAB-OprM pump. In addition, we successfully resolved the crystal structure of NalD protein. The result of structural alignment with TtgR protein in Pseudomonas putida as well as EMSA, ITC and plate assay showed Asnl29 and His 167 are involved in NalD-novobiocin interaction. These results reveal a diversity of regulatory mechanisms in the MexAB-OprM multi-drug efflux, and provide insight in bacterial antibiotic resistance.NalD protein and TetR, the tetercycline-resistance regulatory protein in E.coli, both belong to TetR family of transcriptional regulatory proteins. The result of structural alignment showed that these two proteins are highly similar, sepecially in the DNA-binding domain, In order to reconstruct NalD protein for recognizing TetR’s DNA sequence, the DNA-binding domain of NalD protein is substituted with the corresponding area of TetR protein using protein engineering techniques. The reconstrunted protein was co-expressed with the chaperone plasmid in E.coli BL21 (DE3) strain, and was further purified using FPLC system. The result of EMSA showed that the modified proteins are still active and could recognize the DNA-binding sequence of TetR protein. This suggests that it is feasible to reform proteins’ functions according to their structures.