The Effect Of Asp60 On The Structure And Function Of Myoglobin

To probe the effects of surface-charged residue Asp60 on the structure stability,functions of horse heart myoglobin and its interaction with Cu²⁺,the code of Asp60,was changed into that of Lys by PCR site-directed mutagenesis.The mutant gene was ligated into PstI/BamHI-cut pGYM and the resulting plasmid was transformed into E.coli BL21.The mutant protein was expressed in BL21 successfully.The bacteria containing mutant myoglobin were treated with lysozyme,and then was purified by ammonium sulfate precipitation,ion-exchange chromatography and gel filtration,the recombinant Mb(D60K) was purified with 96%purity.Circular dichroism and UV-vis spectras were employed to monitor the defolding process of wild-type and mutant myoglobins under different temperatures,pH and the concentrations of guanidine hydrochloride.The Tm of protein is decreased from 68.1℃[Mb(WT)]to 66.4℃[Mb(D60K)],(pH)_1/2 of protein is decreased from 4.32[Mb(WT)]to 4.19[Mb(D60K)]and the mid-concentration of guanidine hydrochloride of protein is increased from 1.95mM[Mb(WT)]to 2.10mM[Mb(D60K)]for the protein denature.The results show that the mutant is less stable towards thermal,but more stable towards acid and chemical denaturation than wild-type myoglobin.Spectroscopic methods were used to investigate the interaction between the mutant D60K and Cu²⁺.Data from UV-Vis absorption spectra shows that UV absorption peak of D60K blue-shifted and intensity enhanced,demonstrating that hydrophobic interaction among hydrophobic residues in D60K is decreased.Date from fluorescence spectra shows that 1:1 ratio conjugated compound between D60K and Cu²⁺ is formed,the quenching belongs to static fluorescence quenching,and Mb(D60K) has a higher affinity with Cu²⁺ than that of wild-type myoglobin.The thermodynamic parameters are calculated according to van't Hoff equation,which indicates that static interaction plays major roles in the binding process.The binding distance r is 2.48 nm based on F(o|¨)rster theory of non-radiation energy transfer.Date from synchronous fluorescence spectra shows that Cu²⁺ results in the decrease of hydrophobic effect of tryptophan residue in Mb(D60K).Date from circular dichroism spectra shows that Cu²⁺ has strong impact on Mb(D60K) conformation with the change of the micro-circumstance of aromatic amino residues and decreases theα-helical content of the protein.The Km of D60K for 2-methoxyphenol is 2.19 mM more than 1.26mM of wild type.The optimum temperature and optimum pH for the peroxidase activity of Mb(WT)and Mb(D60K) are at about 60℃and pH5.5 respectively,and the peroxidase activity of Mb(WT) is higher than that of the mutant D60K at the same temperature.