Cytochrome B ₅ Ser64 On The Effects Of Protein Structure And Properties

Cytochrome b5(cyt b5) is an important electron-transfer hemoprotein existing in biological systems,which associates a heme prosthetic group non-covalently.Cyt b₅ is capable of accepting and donating single electron through the change between the ferrous and ferric cation.The membrane-bound microsomal cytochrome b₅,which has a molecular weight mass of～16 kDa and 130 amino acids.The native form of cytochrome Lb₅ can be obtained using lipase,which contains 93 amino acids.In this issue,cyt b₅ all refers to the cytochrome b₅ containing 93 amino adds,Lb₅. Cytochrome b₅ is composed of two independent domains:the heme-containing hydrophilic portion(Core 1) which is functional domain of cytochrome b₅.The heme-holding ability depends mostly on the strong axial ligation provided by residues His63 and His39.the another is a hydrophobic core consisted of nonpolar amino acid (Core 2),maintaining the structural stability of the protein.The NMR and X-ray structures of cytochrome b₅ show that Ser64 locates at the edge of heme-containing hydrophobic pocket.There are two hydrogen bonds formed between Ser64 and heme propionate-7.One of the carboxylate oxygen atoms forms a hydrogen bond with the serine hydroxyl group while the carboxylate oxygen atom forms a hydrogen bond with the Ser-64 main-chain amide group.Set64,heme propionate-6,7,Val61 and Gly62 formed a hydrogen bonds network which stabilizes the heme and keep its conformation.And the same time Ser64 is the N-terminal of the fifth a-helix(Ser64-Phe74),connects the fourth a-helix(Ala54-Va161) through a three amino acids loop(Val61-Gly62-His63).Ser64 could be critical for the stability of the polypeptide.In general,The structural stabilitiy of cytochrome b₅ is associated with two key factors:the stability of heme binding to polypeptide,and the stability of the secondary and tertiary structure of the polypeptide.The polar amino aicds lying in the edge of hydrophobic pocket can affect the statbility mostly.Site-directed mutagentic method plays an important role in studying the effects of specific amino acid in protein structure,function and properties.Temperature-,acid-and detergent-(such as, GdnHC1) induced protein denaturations are typical methods of studying protein stability,folding and unfolding process,which can offer much information about the structural stabilities.UV-visible spectrum(UV-vis),CD(Circular Dichroism) and fluorescence spectrum reflect the disturbance of heme,polypeptide and aromatic amino acids,are also useful in examining structural stability.We made the conserved mutation in cytochrome b₅ S64T,and non-conserved mutations in the cytochrome b₅ S64K,S64N and S64H mutant proteins,in which all substitutes are hydrophilic amino acids.The mutant genes were constructed by PCR, and were expressed in E.coli BL21(DE3)pLysS.The cells were harvested by centrifugation.After sonication of cells and ammonia sulfate precipitation,the mixture solution was subsequently applied on a DEAE DE-52 and Sephadex G-75 and G-25 column to obtain highly purified proteins.SDS-PAGE and Electrospry mass spectroscopy analysis of the four mutant and wild proteins of cytochrome b₅ confirmed the purity and validity.The UV-vis spectrum,CD spectrum,redox potential and stability studies were done to find out the changes of structures and stabilities.We have observed that,in mutant S64T,the disturbances of the overall polypeptide,hydrophobic pocket are quite small.Mutant S64K changed a lot,and mutant S64N and S64H changed distinctly,and changes between this two mutants are similar.The study of wild type cytochrome b₅ and its S64X mutants shows that,for the acid-induced denaturation is to disturb the effect between axial ligations and the heme, the pH_m changed slightly.However,the temperature-and GdnHC1-induced denaturations affect the interactions in the polypeptide seriously,the stabilities of S64X mutant decrease distinctly.And the stability order of this five protein is:Cyt b₅ S64K＜Cyt b₅ S64H≈Cyt b₅ S64N Cyt b₅＜S64T＜Cyt b₅ WT,while S64K mutant protein is the less stable,it may beacause the lysine is so large that disturbance increased.This study results show that,Ser64 is very important for maintaining structure of the fifth a-helix,and could disturb the Core 2,which leads to the decrease of the mutant protein structural stability.