Probe Of The Effect Of Asp44 On The Structure And Function Of Myoglobin

Structural stability and the right folding of proteins are important for their biological functions,and the relationships between structure and function for the proteins have been widely studied by many experts.Among different interactions that contribute to the protein stability(hydrophobic effect,hydrogen bonding,packing interactions,and hydration),the effects of interactions between ionizable groups in the protein surface (charge-charge interactions) attracted little attention until recently.However, in past years, there have been a number of reports showing that charge-charge interactions on the protein surface are capable of modulating protein stability.Myoglobin is one of the most thoroughly studied proteins with respect to structure and function.It has been reported that when the Asp(D)44 on the surface of horse heart myoglobin is mutated to Lys(K),the electron transfer rate between Mb(D44K) and Cytochrome b₅ was increase by ten fold.But there is no report about the influence of this residue on its stability and functions.To probe the effects of surface-charged residue Asp44 on the structure stability and functions of horse heart myoglobin,the code of Asp44,GAT in the gene of horse heart myoglobin was changed into AAA for 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 lysozym.Then the mutant protein was purified by ammonium sulfate precipitation,ion-exchange chromatography and gel filtration,the recombinant Mb(D44K) 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 Mb(D44K) is increased from 71.9℃ to 75.1℃ and (pH)_1/2 of Mb(D44K) is decreased from 3.95 to 3.88.The results show that the mutation of the surface-charged residue Asp44 to Lys44 increases the protein's stability on its resistance to heat and pH.But mid-concentration of guanidine hydrochloride of Mb(D44K) is decreased from 1.88mM to 1.75mM for the protein denature.The Km of D44K for 2-methoxyphenol is 1.9 mM less than 3.3mM of wild type.The optimum temperature for the peroxidase activity of Mb(WT) and Mb(D44K) is 50℃. And Its optimum pH for the peroxidase activity of Mb(D44K) is decreased from 5.5 to 5.0. And the peroxidase activity of Mb(D44K) is higher than that of Mb(WT) at the same...