| Denaturation and aggregation of proteins usually interfere with their normal biological function,ultimately leading to various serious diseases such as neurodegenerative diseases and type Ⅱ diabetes.Although the clinical manifestations of these diseases are different,they are very similar at the molecular level in that abnormal monomeric proteins undergo conformational chan ges under the influence of external factors,which in turn promote the formation of amyloid fibrils.Metal ions(especially transition metal ions)are currently known to be important factors that can affect protein denaturation and aggregation in humans and are therefore of great interest,yet the mechanism of their action in causing amyloid aggregation remains unclear to date.Indeed,most previous studies have focused on the structural features before and after denaturation,and the lack of spectroscopic-based kinetic information has led to partial controversy.Therefore,elucidating the effects of metal ions on protein denaturation and aggregation kinetics is an important prerequisite for a deeper understanding of the ionic mechanism of action and the development of relevant drugs.Numerous experiments have shown that Cd(Ⅱ)ions are concentrated in the plasma,liver and brain tissues of Alzheimer’s disease patients.For this reason,Cd(Ⅱ)ions were selected as the representative of this study,which was devoted to explore the mechanism of the action of Cd(Ⅱ)ions on the denaturation and aggregation of egg white lysozyme(HEWL),hoping to provide valuable information for revealing the molecular mechanism of protein aggregation in related diseases.In this work,we systematically investigated the denaturation and aggregation kinetics of lysozyme under the action of Cd(Ⅱ)ions and the synergistic effect of Cd(Ⅱ)ions and acids by using Raman spectroscopy as the main technique and combining the research methods of thioflavinT(ThT)fluorescence,far-UV circular dichroism(CD)spectroscopy,transmittance analysis and atomic force microscopy(AFM),and explored the evolution mechanism of the secondary and tertiary structures of the related proteins.(1)Mechanism of Cd(Ⅱ)ion-induced "inside-out" denaturation of HEWL.We measured the kinetic changes of the tertiary and secondary structures of HEWL in the presence of Cd(Ⅱ)ions using Raman spectroscopy in combination with ThT fluorescence spectroscopy and far-UV CD spectroscopy under thermal incubation conditions to investigate the effect of Cd(Ⅱ)ions on the denaturation kinetics of HEWL.The results showed that the Cd(Ⅱ)ions entering the molecular cavity first bound to the a-helix and triggered its unraveling,and this internal structural change led to the"collapse" of the molecular conformation of HEWL,which resulted in the exposure of internal hydrophobic residues,i.e.,the unfolding of tertiary structure.During the whole denaturation process,the internal secondary structure of the protein is changed simultaneously,resulting in the change of the overall conformation of the molecule,thus showing a typical "inside-out" denaturation mechanism.Under the action of Cd(Ⅱ),disordered aggregates with a more disordered tertiary structure and some residual αhelix structures were formed.This study will provide valuable information for further understanding the molecular mechanism of ion-specific effects in protein structure and the mechanism of toxicity of exogenous ions.(2)Mechanism of the role of Cd(Ⅱ)ions in the aggregation of HEWL under thermal/acidic conditions.Under thermal/acidic conditions,we investigated the kinetic processes of tertiary and secondary structural transformations of HEWL at the molecular level by Raman spectroscopy,ThT fluorescence spectroscopy and far-UV CD spectroscopy.In addition,AFM imaging and transmittance measurements were applied to observe the morphological characteristics and formation kinetics of the aggregates.By comparing the experimental results of both conditions in the presence and absence of Cd(Ⅱ)ions,we found that Cd(Ⅱ)ions can effectively accelerate the disruption of tertiary structures and promote the direct conversion of a-helix structures into organized β-sheet structures by skipping the intermediate randomly coiled structures.Throughout the protein aggregation process,Cd(Ⅱ)ions prefer to induce the assembly of initially formed oligomers into larger disordered aggregates,such as gels,rather than amyloid fibrils.The "off-pathway" aggregation pathway is dominant in this process.Our study not only has important implications for understanding the relationship between metal ions and protein conformational diseases,but also advances the understanding of the corresponding ion-specific effects. |
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