Structure, Solution Property And Drug Target Studies Of Intein

This dissertation contributes to the mechanism studies of intein on the basis of protein structure and solution property, and the potential drug target of intein for anti-Mycobacterium tuberculosis. These studies cover the following three parts:1) Solution structures and dynamics of Mycobacterium tuberculosis RecA mini-inteins. The protein structure solved NMR in this work shows that the mutation of key residues have no apparent effect on protein structures, although these mutations really influence the splicing activity. The solution structures solved in this work are similar to the crystal structure in literature. However, NMR analyses indicate that the structure stability can be enhanced by V67L mutation. Furthermore, we also detected that D121plays an important role in protein splicing and C-terminal cleavage activities.2) Detailed investigation of interaction of two splicing domains of DnaE split intein from Synechocystis sp. PCC6803. We discovered that the two domains are intrinsically disordered individually, while synergistic protein folding occurs along with two domains interaction. The synergistic folding of two splicing domains are characterized using CD spectroscopy, fluorescence spectroscopy, gel filtration, DLS, SPR and NMR.3) Potential anti-tuberculosis drugs target of intein and its inhibition by metal complexes. Our results indicated that the inhibition of intein can effectively prevent the proliferation of Mycobacterium tuberculosis cells. Platinum complexes exhibit better inhibition efficiency than other metal complexes, and the structure/activity relationship has been concluded in this assay. Among all compounds analyzed, an antitumor drug, cisplatin demonstrated the best inhibition efficiency.In chapter1, we provide a brief review of intein and protein splicing, which covers the distribution of intein, gene mobility, the conservation motif of intein, the type of intein, the mechanism of protein splicing, the structure of intein and the application of intein. Protein splicing is further extended in the following chapter on two classes:1) cis-splicing is named after cis-intein whose gene is integrity,2) trans-splicing is named after trans-intein whose gene is split.In chapter2, we have solved the solution structures and determined dynamic properties of M. tuberculosis RecA mini-inteins. A comparison between the backbone structure of crystal and solution structures shows that there is no apparent difference, namely, the structure is also a typical HINT module composed mainly of β-strand secondary structures. Hydrogen/Deuterium exchange have been employed to validate the stability of RecA intein can be enhanced by V67L mutation, while the X-ray structure show only negligible difference caused by V67L mutation. Moreover, D121G mutation suppresses the N-terminal cleavage activity and produces a faster C-cleavage mutant CM. Based on H/D exchange and chemical shift mapping, NMR studies reveal that D121G mutation eradicates H-bonding network constructed by D121. The C-terminal cleavage can happen individually without the participation of N-terminal and the CM has even faster C-cleavage without the restrain of H-bond. It can be concluded that by the linkage to the C-and N-termini, D121can enhance protein splicing, however, it impedes the individual cleavage on either N-or C-terminal.In chapter3, detailed investigation has been performed to describe properties of Synechocystis sp. PCC6803DnaE split intein in solution and interaction between the two split splicing domain using CD spectroscopy, fluorescence spectroscopy, gel filtration, DLS, SPR and NMR. The process of protein trans-splicing involves an initial binding interaction between the two split inteins followed by a four-step-reaction splicing mechanism, so the process of molecular recognition and binding must influence the splicing activity and efficiency of trans-intein. In order to understand the processes of interaction, we have expressed and purified two splicing domains, and complex of DnaE-N123-C36. Circular dichroism spectra demonstrate the structure of DnaE-N123is almost disordered, but CD studies also show that some residual structure of DnaE-N123gradually disappears with increasing concentration of urea or guanidinium chloride. Further, the fact of intrinsically disordered N-terminal domain is confirmed using circular dichroism spectra, gel filtration and DSL. The results show that the N-terminal domain can be shifted to order region by adding C-terminal domain using bioinformatics analysis, as well as the following experiments. In order to confirm the interaction of the two partners, the association constant of the two partner was determined using SPR and ITC. HSQC spectra suggest that the folding of DnaE-N123can be induced by adding the C-terminal domain. In summary, the conformation change of N-terminal domain induced by the C-terminal domain demonstrates that in the process of trans splicing, it requires protein recognition, binding and folding. This process should depend on the solution condition, thus the in vitro protein splicing efficiency can be controlled. In chapter4, we have examined the inhibition of protein splicing as a novel strategy for new development of antitubercular chemotherapy. Our results demonstrate that cisplatin effectively inhibit the protein splicing both in vitro and in vivo screen system, furthermore, The growth of M. tuberculosis H37Rv is significantly inhibit by cisplatin. ESI-MS, NMR and mutation analysis show that the inhibition of cisplatin is contributed by cysteine of the first residue of intein。In summary, this study suggests that cisplatin is a potential anti-tuberculosis agent and inteins are potential therapeutic targets for tuberculosis therapy.