Neurotoxic species of misfolded hSOD1 are involved in the process of causing amyotrophic lateral sclerosis (ALS), which is a devastating neurodegenerative disease. Considerable evidence exists on that hSOD1 mutants-mediated toxicity is resulted from gain-of-function, while the mechanism of this toxicity is unknown yet. In the present study, we focused on the possible mechanism of two point-mutations (namely L67P and D76Y) on metal-binding sites and their possible consequent effects on ALS progress. For this purpose, the exposed hydrophobic patches were detected using ANS fluorescence and the formation of hSOD1 aggregates was monitored using the ThT fluorescence and absorption spectra of Congo red (CR). Moreover, to assess aggregate's morphology, transmission electron microscopy (TEM) was used. The specific activity of wild-type, as well as L67P and D76Y mutants was obtained as 12345, 8625, and 7066 U/mg, respectively. The existence of b-sheet-dominated structures was observed under amyloidogenic conditions using far UV CD and FTIR spectroscopy. As well, comparative study of wildtype and mutants by intrinsic and extrinsic fluorescence revealed structural alterations and the increased hydrophobic surface pockets, respectively. The formation of the amyloid fibrils was monitored under destabilizing. The results of ThT and CR showed the process formation of amyloid aggregates and moreover, the presence of morphological forms was confirmed by the TEM image. Overall, our findings supported that mutation in the zinc-binding loop could significantly increase the tendency to mediate amyloid aggregation and it may possibly trigger misfolding and fibrillar aggregation, which are pathological changes in familial forms of ALS.
