The Inhibition Of Metal Ions On Protein Splicing And XAFS Study Of Coordination Structure Of Cu(His)₂ Aqueous Solution

In this dissertation, we study several aspects about protein splicing in Mycobacterium tuberculosis RecA intein. First of all, we made a brief review about protein splicing and the internal protein (intein). It includes the definition of protein splicing, the mechanism of protein splicing, the critical amino acids in protein spling, the nomenclature of intein, inteins'distribution, the classification of inteins, the conserved amino acids and motifs in intein, and the application of inteins.We screen several protein splicing inhibitors, using both in vivo and in vitro screening reporter system. The in vivo reporter system are TS reporter system and ALSII reporter system, the in vitro reporter system is GFP-RecA intein reporter system. The results for complex P-0810 is that the IC₅₀ in vitro is 2.5μM, while the inhibition of inteins plicing using TS reporter system has the IC₅₀ of 7.71μM and in ALSII reporter system the IC₅₀ is 18.47μM. For the in vitro system we used GSH to consume complex P-0810, and the inhibition can be attenuated. So we concluded that the inhibition of splicing is reversible.In order to get a better understanding of metal-protein interaction, we planned to do x-ray diffraction. So single-crystals of metal-protein are needed. Three proteins were expressed and purified using metal chromatography column followed by size-exclusive chromatography. The buffer pH, salts'concentration and precipitants'concentration are optimized to acquire single-crystals. At present, the optimization process is still going on.In a previois study took in our lab, we discovered that the His73 in intein might coordinate with copper ions and play a role in inhibiting the protein splicing. So we studied the coordination mode of Cu(His)₂ solution using XAFS method. A further significance of this study is that Cu(His)₂ plays an important role in copper ions balance in human blood by exchanging copper ions with albumin. From our XAFS results, we can conclude the coordination mode under pH 2.8, 4.5, 7.4 and 9.8.