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Abstract
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Familial amyotrophic lateral sclerosis (fALS) is a neurodegenerative disorder; approximately 20% are caused by dominant mutations in the gene encoding Cu/Zn human superoxide dismutase (hSOD1). To date, several mutations have been iden- tified and linked to ALS. This study was designed to evaluate the effect of two ALS-associated point mutations, namely L106P and L106F, located in loop VI (Greek-key loop). Our analysis was performed through multiple algorithms on the structural characterization of hSOD1 protein using experimental studies, computational predictions, and molecular dynam- ics (MD) simulations. Results from single-nucleotide polymorphisms (SNPs) predicted the deleterious and destabilizing effect of mutants hSOD1. MD outcomes also showed that the flexibility and stability of mutants were reduced compared to the wild-type. Besides, analysis of the gyration radius indicated higher compactness for mutants, suggesting that replacing amino acid at the Greek key loop can alter the protein compactness and hydrophobicity compared to the WT. The specific activity of WT-hSOD1, L106F, and L106P was obtained as 7031, 2812, and 1942 U/mg, respectively. Comparative results of WT-hSOD1 and mutants by intrinsic, ANS, and quenching fluorescence revealed structural compactness and increased hydrophobic surface pockets. Overall, our findings supported that mutation in loop VI could significantly increase the ten- dency to mediate trigger protein misfolding and aggregation in vitro, leading to decreased stability and loss of function of the mutated protein. Hence, such mutations' results are possibly a prerequisite for a better understanding of fALS pathogenicity.
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