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Abstract
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Misfolding and accumulation of Cu/Zn-superoxide dismutase (SOD1) and the formation of amyloid fibrils is one of the hallmarks of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). This research aims to explore the structural characteristics of the R115G mutant in the presence and absence of the ionic liquid (IL) known as 1-butyl 3-methylimidazolium thiocyanate. In this study, the plasmid containing the R115G mutation was transformed into E. coli BL21, induced with IPTG and purified using nickel-agarose affinity chromatography. The protein purity was confirmed using SDS-PAGE. For structural studies, the protein was dialyzed and the fluorescence intensity was determined at concentrations of 0.1 and 0.05 mg/ml for intrinsic and extrinsic fluorescence, respectively. To assess the effect of IL on the structure of the R115G mutant, concentrations ranging from 0-100 μM were utilized. As IL concentration increased, fluorescence intensity decreased, indicating a shift towards a more non-polar environment surrounding tryptophan residues. Extrinsic fluorescence with ANS marker was employed to study the hydrophobic pocket of the protein. While no significant difference was observed in the fluorescence intensity with or without IL and without incubation time, an increase in incubation time up to 12 hours led to heightened fluorescence intensity and exposure of more hydrophobic envelopes. Overall, the findings suggest that the presence of ionic liquids can influence the structural characteristics of the SOD1 enzyme, potentially aiding in the regeneration and prevention of amyloid fibril formation.
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