NMR Studies On New Delhi Metallo-beta-lactamase(NDM-1) And The Expression And Purification Of Japanese Encephalitis Virus Capsid Protein

With the widely use of antibiotics,the problem of bacterial resistant antibiotic becomes more and more serious.β-lactamases which is expressed by the resistant antibiotic bacteria are attracting much attention.By 2009,a novel resistance factor called the New Delhi metallo-β-lactamase-1（NDM-1）has been found to hydrolyze nearly almost all β-lactam antibiotics except polymyxin and tigecycline,which has had an enormous impact on human health clinically.Structural biologists have studied about the structures on the powerful ability of antibiotic hydrolysis of NDM-1.But the detailed molecular mechanism is still not clear.In the thesis,we use conventional NMR and ¹⁹F NMR to study about the solution structure and the molecular mechanism of NDM-1.Firstly,we constructed the plasmid,expressed and purified the protein samples for solution NMR experiments,including 3FY-labeled,5FW-labeled and uniform 2H/13C/15N-labeled NDM-1.Triple resonance NMR spectra of uniform 2H/13C/15N labeled di-Zn-NDM-1 were acquired and processed,and the assignments of the main chain of NDM-1 were completed.Using TALOS+ software,the secondary structure prediction was obtained,which indicated the secondary secondary of NDM-1 in solution was identical with that in crystal state,both have the same 5 a-helix,12β-sheets and loops.The result can help us to get the information of the dynamics of NDM-1 later using NMR methods.Meanwhile,using ¹⁹F NMR,the conformational changes of NDM-1 at different functional states,including apo-NDM-1（non-active）,di-Zn-NDM-1（active）,and L-Captopril-bound-NDM-1（deactive）,can be detected sensitively and robustly.Zn2+titration experiments indicated the loops around the active site exsist slow-timescale conformational exchange when NDM-1 binds Zn2+ ions.It means Zn2+ ions may not only participate in the hydrolsis reaction,but also regulate the conformation exchange around the active site to facilitate the binding of antibiotics and the release of hydrolysates during catalysis reaction.Besides,we introduced the method to overexpress Japanese encephalitis virus capsid protein（JEV-C）.We constructed the capsid-expressed plasmid,expressed and purified the capsid protein samples for solution NMR experiments,which lacked the hydrophobic fragment（residues W114-A127）in C-terminal.The preparation of high purity sample will be the foundation to get the NMR structure and understand more functions of capsid protein in virus particles.