Structural and conformational studies of loop VI in copper–zinc superoxide dismutase by site-directed mutagenesis

Backgrounds: The human superoxide dismutase (hSOD۱) is an antioxidant enzyme thatcatalyzes two molecules of superoxide anion into hydrogen peroxide and water. More than ۱۸۰different mutations in the SOD۱ gene have been reported in association with amyotrophic lateralsclerosis (ALS), many of which are due to amino acid replacement. In this study, by substitutingleucine to proline (L۱۰۶P) at position ۱۰۶, the effect of mutation on activity and structuralproperties was investigated.Materials and Methods: The vector pET-۲۸a-hSOD۱ was used for mutagenesis by QuickchangePCR methods. The WT- hSOD۱ and mutant protein was expressed in E. coli-BL۲۱(DE۳), at ۲۲ °C, ۲۳۰ rpm for ۱۸h, and purified using Ni-NTA agarose affinity chromatography.Comparative structural study of wild type and mutant was performed by intrinsic and extrinsicfluorescence.Results: The results of bioinformatics servers showed the destructive effect of mutations onprotein structure. The specific activity of wild-type and L۱۰۶P mutant was ۷۰۳۱.۲۵ and ۱۹۴۲.۸U/mg at a concentration of ۰.۱ mM pyrogallol, respectively. Increased of mutant intrinsicfluorescence compared to wild-type indicates structural changes and tryptophan exposure in anon-polar environment. The increased ANS fluorescence mutant compared to wild-type,indicating increase the hydrophobic surfaces in the protein.Conclusion: Overall, our results showed that mutations in loop VI lead to structural changes andimplications for initiation of ALS. Further studies on loop VI provided clearer evidence for thepathogenicity and its association with the ALS phenotype.

کلیدواژه ها:

Human superoxide dismutase ۱ ، Amyotrophic lateral sclerosis ، loop VI ، ANS fluorescence