Screening apo-SOD1 conformation stabilizers from natural flavanones using native ion mobility mass spectrometry and fluorescence spectroscopy methods.

Abstract

A large number of studies have shown that the production of aberrant and deleterious copper zinc superoxide dismutase (SOD1) species is closely related to amyotrophic lateral sclerosis (ALS). Therefore, it is of great significance to screen effective inhibitors of misfolding and aggregation of SOD1 for treating ALS disease. The interaction between flavanone compounds with apo-SOD1was investigated using native electrospray ion mobility mass spectrometry (native ESI-IM-MS). Binding affinities of ligands were compared using native MS, ESI-MS/MS, collision-induced unfolding, and competitive experiments. The effect of ligands on apo-SOD1 aggregation was investigated using the fluorescence spectroscopy method. The results of MS showed that the binding affinity of liquiritin apioside was the strongest, better than the corresponding monosaccharide and aglycone, indicating that the presence and the number of glycosyl group are beneficial to enhance ligand affinity to protein. The results of fluorescence spectroscopy for inhibiting protein aggregation in vitro were consistent with the binding affinity. In addition, the results of the collision-induced unfolding indicated that liquiritin apioside can slow down the unfolding of the protein. Meanwhile, the results of competition experiment suggested that liquiritin apiosides share different binding sites with naringin and 5-fluorouridine, which are significant for the structural stability of SOD1. This study revealed that the binding of liquiritin apioside can stabilize apo-SOD1 dimer and inhibit the aggregation of apo-SOD1, and illustrated that native ESI-IM-MS is a powerful tool for providing insight into investigating the structure-activity relationship between small molecules and protein, and screening protein conformation stabilizers.