The Purification And Structure Analysis Of Tc And Tb Inorganic Pyrophosphatase

Trypanosomiasis are parasitic diseases caused by trypanosomes,which are widely popular in the Sub-Saharan Africa and America.Trypanosomiasis has caused the serious public health problem and brought huge economy loss.Currently,the limited chemotherapeutic agents have strict treatment protocols,frequent toxic side effects and rising drug resistance.The develoment of new anti-trypanosomatid drugs is extremely urgent.VSP is a soluble inorganic pyrophosphatase located in the acidocalcisome of trypanosomes.It is essential for growth of bloodstream forms in mammalian host and hence is an promising target for Trypanosomiasis.Now all of the catalytic mechanism,the function of N-terminal domain,the structures of VSP,and the inhibitor structures are poorly understood.So the study of VSP loacted in the trypanosomes is crucial.In this study,soluble pyrophosphatase derived from Trypanosoma cruzi and T.brucei,by Ni column and DEAE column chromatography to obtained the high purity of TcVSP protein,?N1-TcVSP and TbVSP protein.Crystal structures of TcVSP-BPH1260,PPi-?N1-TcVSP,MgSO4-?N1-TcVSP and TbVSP were obtained by X-ray crystal diffraction technique.The T.cruzi and T.brucei proteins form a tetramer both in the solid-state and in solution.However,in both proteins there is head-to-tail dimer packing in which the EF-hand domain in one chain interacts with the PPase domain in a second chain.The protein structure is the first of the soluble phosphatase family I contains two domain structure of protein.Furthermore,the molecular size exclusion chromatography with Muti-angle light scattering detection(SEC-MALS)was used to study the influences of the N-terminal domain of TcVSP and TbVSP on the molecular aggregation state.The results showed that the trypanosome VSP have similar N-terminal calmodulin domain(EF-hand domain),yet a functional diversity.TbVSP,in the absence of calcium ion,is a variety of polymer mixtures.TbVSP,when the presence of calcium ions,is the formation of octamers.However,the molecular aggregation state of TcVSP has nothing to do with calcium ions.Although the trypanosome VSP have similar N-terminal calmodulin structure domain(EF-hand domain),its functional diversity.This study prepared a protocol for the expression and purification of the inorganic pyrophosphatase and obtained the crystal structures of trypanosoma pyrophosphatases,which will elucidate the relationship between the structure and function of trypanosoma VSP and help develop novel chemical drugs for trypanosomiasis treatment.