Effect Of Manganese Ions On Amyloid Kinetics Of Hen Egg White Lysozyme By Raman Spectroscopy

The formation of amyloid fibrils from misfolded proteins is thought to be a hallmark of several neurodegenerative diseases.The production of these amyloid fibrils is closely related to environmental factors,(e.g.pH,temperature and various metal ions).Among these environmental factors,metal ions are considered to be one of the important factors causing neurodegenerative diseases due to their ability to specifically bind polypeptides.Therefore,understanding the role of metal ions in protein fibrosis is important for understanding the development of related diseases as well as their treatment.In recent years,there is increasing evidence that manganese ions(Mn2+)may be one of the risk factors for protein amyloid fibrillation,however,previous studies have focused on the morphological changes before and after protein denaturation and the macroscopic structural observations of the formation of aggregates,while the microscopic mechanism1 of the role of Mn2+ in its denaturation process are still not well elucidated.In this paper,we investigated the specific role of Mn2+in the denaturation of hen egg white lysozyme using Raman spectroscopy,circular dichroism spectroscopy(CD spectroscopy),atomic force microscopy(AFM),ThT fluorescence spectroscopy,and ultraviolet-visible absorption spectroscopy(UVVis).First,we investigated the effect of Mn2+ ions when acting alone on the thermal denaturation(65℃)of lysozyme protein.The results of Raman and CD spectra clearly showed that the presence of Mn2+ alone did not significantly affect the secondary structure of lysozyme,but significantly promoted the unfolding of the tertiary structure.Subsequently,we further investigated the effect of Mn2+ on the kinetics of lysozyme fibrillation under thermal(65℃)/acidic(pH=2.0)conditions,examining the competitive and synergistic effects of metal ions and protons.By observing the ratio of the half-height full width of the tryptophan residue at the 759 cm-1 peak and the intensity of the peaks at 1340 cm-1 and 1360 cm-1 in the Raman spectra,we confirmed that,relative to protons,Mn2+ is able to induce the unfolding of the tertiary structure of lysozyme more efficiently,thus facilitating the transition from protein monomers to oligomers.AFM images and UV-Vis absorption spectra,as well as the evolutionary kinetics of the two Raman spectral pointers mentioned above,clearly indicate that Mn2+ promotes the formation of amorphous aggregates rather than amyloid fibrils.Furthermore,the N-Cα-C intensity at 933 cm-1 and amide I position of the Raman spectrum and the ThT fluorescence experiments show that Mn2+ also plays a significantly accelerating role in the transition of the secondary structure from α-helix to organized β-sheet structure,which can be attributed to its synergistic effect with proton action.In conclusion,we can state with certainty that Mn2+ contributes more significantly to the development of amorphous aggregates.This is a trustworthy hint to comprehend the information about how excessive Mn exposure causes neurological illnesses.