Nickel Ion-induced Aggregation And Amyloid Fibrillation Kinetics Of Hen Egg White Lysozyme By Raman Spectroscopy

Protein amyloid fibrillation is a misfolding process that forms specific fibrillar aggregates rich in organized β-sheet structure.It is considered to be a hallmark of several major human diseases,such as Alzheimer’s disease(AD)and Parkinson’s disease(PD).Therefore,the formation mechanism and influencing factors of amyloid fibrillation have received a lot of attention,which is expected to provide an important reference for the treatment or remission,or even prevention of related diseases.Among the many external influencing factors,abnormal concentrations of metal ions,which are essential enablers of numerous vital metabolic processes,may also act as environmental toxins to disrupt normal physiological activities and induce protein amyloid fibrillation.However,the microscopic mechanisms of metal ion regulation of protein amyloid fibrillation processes have not been well studied at the molecular level so far.Therefore,it is of great scientific and application value to study the effects of metal ions on protein aggregation kinetics and to reveal the mechanism of their effects on protein aggregation.In this thesis,the effect of nickel ions and others on protein aggregation was investigated in depth using hen egg-white lysozyme(HEWL)as a model protein,focusing on Raman spectroscopy and combining various spectroscopic characterization methods.The main research contents and results are as follows:(1)Nickel and magnesium ions have specific effects on aggregation of lysozyme.Under thermal incubation(65℃)conditions,we explored the molecular structural changes of HEWL aqueous solutions after longer(196 h)incubation in the presence of Ni(Ⅱ)or Mg(Ⅱ)ions,respectively,which is detected by four Raman characteristic peaks of spontaneous Raman spectroscopy.The four spectral pointers are the N-Ca-C stretching vibrational band at 933 cm-1 and the amide I band at 1600-1700 cm-1,which are closely related to the secondary structure of the protein,and the tryptophan residue bands at 759 cm-1 and 1340-1360 cm-1,which are associated with the tertiary structure.The experimental results showed that Ni(II)ions can significantly promote the unfolding of the tertiary structure of the protein after 196 h of thermal incubation,but have no significant effect on the secondary structure transition.Moreover,this acceleration exhibits a significant positive correlation property with the concentration of metal ion.On the contrary,the same concentration of Mg(Ⅱ)ions affects the aggregation by retarding the secondary structure transition.Thus,this strongly suggests that metal ions have specific effects on protein aggregation and that electrostatic interactions play an important role in the protein aggregation process.The present study provides significant information on the interaction between metal ions and proteins in the aqueous environment.(2)Nickel ions change the denaturation pathway of lysozyme in the amyloid fibrillation process under thermal and acidic conditions,which not only accelerates the transition from α-helix to β-sheet,but also promotes the generation of disordered structures more significantly relative to the formation of fibrillar aggregates.We investigated the effect of Ni(Ⅱ)ions on the kinetics of HEWL amyloid fibrillation under thermal and acidic(65℃,pH 2.0)conditions using various spectroscopic experimental techniques such as Raman spectroscopy,thioflavin-T(ThT)fluorescence spectroscopy,intrinsic fluorescence spectroscopy,ultraviolet-visible(UV-Vis)absorption spectroscopy,atomic force microscopy(AFM)imaging,and circular dichroism(CD)spectroscopy.The experimental results showed that in addition to a small accelerating effect on the tertiary structure unfolding of proteins,Ni(Ⅱ)ions have a significant effect on the secondary structure transition,especially skipping the formation of random coils intermediates and promoting the transition of a-helix directly to β-sheet.Furthermore,by fitting analysis of the amide I bands and other characterization methods such as AFM morphological observation,we clearly revealed that Ni(Ⅱ)ions have an inhibitory effect on the formation of amyloid fibrils rich in organized β-sheet structures,and prefer to promote protein assembly into aggregates with predominantly disordered structures,which may be closely related to the binding sites of Ni(Ⅱ)ions and HEWL.The present study provides a wealth of information about specific metal-mediated protein fibrillation processes.In this thesis,the regulatory effects of metal ions on protein amyloid fibrillation have been investigated at the molecular level using a combination of techniques.This deepens the knowledge and understanding of the specific effects of metal ions,and hopefully will provide implications for the prevention and treatment of diseases related to protein fibrosis.