Thermostability Of Manganese Superoxide Dismutase Analysis Designed Via Molecular Dynamics Simulation

Superoxide dismutase?SOD?is an important oxygen free radical scavenger in organism.It disproportionates superoxide anion radicals that produce excess and destruction of cells through the disproportionation reaction to produce H₂O₂ and O₂,plays an important role in the organism to resist oxidative damage.Mn-SOD is one of the SOD protein mainly located in the mitochondria,because the mitochondrial electron transfer chain is the main source of superoxide,approximately 5%of the molecules of O₂ are converted into superoxide during the delivery process,so Mn-SOD is regarded as the first-line defense against oxidative pressure,the research of it has been a hot spot in frontier areas.But the stability of Mn-SOD in body is bad and the half-life is short,so that its function is limited in a certain extent.In order to improve the thermal stability of Mn-SOD protein,the computer aided design software Discovery Studio 4.0?DS 4.0?was used to modify the Mn-SOD protein to improve its thermal stability.The main research contents and experimental results are as follows:1.The complete three-dimensional structural model of mouse Mn-SOD was constructed by homology modeling with DS 4.0 software using the template of three-dimensional structure of human Mn-SOD protein?PDB accession number:2ADQ?.Ramachandran plot and Verify-3D evaluation shows that the structure model of Mnsod.M0014 is reasonable and the overall compatibility are the most credible.2.The amino acid sequence of Mn-SOD from Homo sapiens?Bos tanrus?Equus caballus?Oryctolagus cuniculus and other species were received from NCBI nucleic acid database.The amino acid sequence of the above amino acid sequence was matched with Mus Musculus sequence by software DS 4.0,we got the non-conserved amino acids of mouse Mn-SOD protein as the sites to be mutated.3.The three-dimensional structure model of mouse Mn-SOD was loaded into DS 4.0software,virtual amino acid site-directed mutagenesis was used to mutate the non-conserved amino acid residues into other 19 amino acids.We finally got single,double,triple mutants can enhanced the stability of target protein based on the unfolding free energy???Gmut?.4.The wild-type Mn-SOD and the best 15 mutants were subjected to 10 ns molecular dynamics simulation.RMSD,RMSF and Potential energy of wild-type and mutant Mn-SOD protein were analyzed by Analysis Trajectory module.We finally got the best mutated combinations can enhanced the stability of target protein are:single point mutation?SM-MnSOD?:E215W;double point mutation?DM-MnSOD?:V140L,E215W;triple point mutation?TM-MnSOD?:V140L,E155W,E215W.5.The gene encoding Mn-SOD protein about 670 bp was cloned from mouse blood by RT-PCR and PCR amplification,the target mutation gene was obtained by Overlap Extension PCR.We obtained the recombinant plasmid pET-MnSOD and the three mutated recombinant plasmids Mut-pET-MnSOD via digestion,ligation and transformation.6.The wild-type and mutant recombinant plasmids were transformed into E.coli BL21?DE3?for expression,the induced temperature was 18^oC and the final concentration of IPTG was0.5 mmol for 20 h.After sonicated and SDS-PAGE electrophoresis,the results showed that there was a specific target protein band at 28 kD and a large amount of soluble protein in the supernatant.Then the wild and mutated target proteins were purified by Ni-NTA affinity chromatography.7.The enzyme activity of Mn-SOD protein was determined by the improved pyrogallol autoxidation method.The results showed that the specific activity of wild type Mn-SOD protein was 1635U/mg,the SM-MnSOD protein was 1650 U/mg,DM-MnSOD protein was 1795U/mg,TM-MnSOD protein was 2050 U/mg.Among them,DM-MnSOD and TM-MnSOD were increased 9.8%and 25.4%respectively compared with the enzyme activities of wild type MnSOD,while the changes of enzyme activities in SM-MnSOD was not significant.8.The wild-type and mutant Mn-SOD proteins with the same enzyme activity were analyzed for thermal stability,DM-MnSOD and TM-MnSOD proteins have good thermal stability and acid-base resistance compared with wild type Mn-SOD.This experiment provides a calculation idea and method for improving thermostability of protein by computer aided design software,and provides a theoretical basis for designing more new enzymes that can play an important role in high temperature.