Kinetic Insights into Zn²⁺-Induced Amyloid β-Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy

Zn²⁺ has remarkable impacts on amyloid β-protein (Aβ) aggregation, which is crucial in the pathology of Alzheimer's disease. However, the Zn²⁺ concentration in human cerebrospinal fluid is commonly too low for interaction with Aβ, and only during neurotransmission is there a transient release of a high concentration of Zn²⁺. It is difficult to explore the details of the interaction between Zn²⁺ and Aβ within such a short time scale by using ordinary analytical methods. Herein, stopped-flow fluorescence spectroscopy was used to study the fast aggregation kinetics of Aβ₄₂ in the presence of Zn²⁺ in the time scale from 1 ms to seconds. It was found that Zn²⁺ bound to Aβ₄₂ within 1 ms; caused immediate conformational transition around Tyr10, which led to the enhancement of Aβ₄₂ hydrophobicity; and then promoted fast aggregation of Aβ₄₂ through enhanced hydrophobic interactions. Among the two Zn²⁺-binding sites on Aβ₄₂ (K_D = 107 nM and 5.2 μM), the first one of higher affinity had a greater impact on the aggregation of Aβ₄₂. Three kinetic phases were observed in the Zn²⁺-induced fast aggregation of Aβ₄₂, and the fast phase was extremely accelerated by Zn²⁺, indicating that accelerated aggregation mainly occurred in the fast phase. The reactions occurring in this phase were closely related to the association of Zn²⁺ and Aβ₄₂. Moreover, Zn²⁺ largely broadened the pH range of Aβ₄₂ fast aggregation from pH 5.2-6.2 without Zn²⁺ to pH 5.2-7.8 in the presence of Zn²⁺. Besides, the promoting effect of Zn²⁺ on Aβ₄₂ fast aggregation peaked at pH 6.8-7.8, which includes the pH values of the cerebrospinal fluid (pH 7.3) and hippocampus (pH 7.15-7.35). The findings demonstrate the significant effect of Zn²⁺ on Aβ aggregation and provide new insight into its mechanisms.