The Purification And Properties Analysis Of YpHmuT

Yersinia pestis HmuT（YpHmuT）,a hemim protein from Yersinia pestis,belongs to the third class of periplasmic binding proteins.The molecular weight is 29.57 kD.YpHmuT is an important part of bacterial uptake of heme.In Y.pestis,YpHmuT can transfer the bound heme to the heme transporter HmuUV,which in turn transports the heme into the cytoplasm.Previous studies described the crystal structure of YpHmuT before and after binding to hemin.The superposition of the crystal structure shows that the two conformations of proteins（apoYpHmuT and hemin-YpHmuT）combining heme undergo a weak change in the conformation around the hemim binding site.When combined with hemim,the N-and C-terminal of YpHmuT undergo a certain degree of torsion.And the change of the N-terminal domain is mainly the rotation of theα-helix.However,theα-helix,β-sheet,and loop regions of the C-terminal domain all showed a certain torsion.Surprisingly,YpHmuT binds two hemin via tyrosine and histidine,while other periplasmic hemin-binding proteins（such as ShuT and PhuT）can only bind one hemoglobin.Based on the Previous research,the following series of researches on YpHnuT were conducted:Firstly,this experiment proved that YpHmuT belongs to protein inclusion body.We used urea as denaturant to denature the protein and obtain through dialysis and AKTAprime^TMM plus protein purification obtain YpHmuT and YpHmuT-hemin complexes.The purified protein was confirmed by polyacrylamide gel electrophoresis.And the purity reached the spectral analysis requirements.Secondly,the interactions between YpHmuT and hemin/heme were studied by fluorescence spectroscopy,Native-PAGE,electrochemical method,EPR and UV-vis spectra and so on.The results show that the molar ratio of YpHmuT to hemin or heme was 1:2.Iron in hemin or heme interact with His and Try in YpHmuT it was five-coordinate high spins.During the interaction between protein and hemin,the behavior of the hemin/heme-YpHmuT redox couple on the electrode is controlled by the diffusion current.In the process,the diffusion coefficient D was2.267×10^-6cm²·s^-1,the electron transfer rate constant k_s was 1.86×10^-3s^-1,and the electron transfer coefficientαwas 0.25.Finally,the conformational effect of Hemin and Heme on YpHmuT was detected using the hydrophobic probe TNS（2-p-toluidinylnaphthalene-6-sulfonate）.The results showed that,regardless of the presence of hemin/heme,the ratio of YpHmuT to TNS was 1:4,and the binding constant was about 10⁴ M^-1.The distances of TNS with YpHmuT,YpHmuT-hemin,hemin₂-YpHmuT,and heme₂-YpHmuT were 1.4 nm,4.6 nm,5.14 nm,and 1.8 nm,respectively.The addition of hemin/heme causes changes in the conformation of the protein and the conformational change is related to the concentration of the hemin/heme.When the ratio of hemin/heme to YpHmuT is 2,the conformation of YpHmuT was no change.Compared to the same concentration of hemin and heme,the conformational change of YpHmuT induced by hemin is greater.