1. Investigation on the aggregation process of amyloid-β-(16-22) peptides and the dissolution of intermediate aggregates
Dongdong Lin, Yin Luo, Shan Wu, Qianqian Ma, Guanghong Wei and Xinju Yang*
State Key Laboratory of Surface Physics and Department of Physics, Fudan University, Shanghai , P. R. China
The.
Introduction
The aggregation processes of amyloid--(16-22) peptides (A16-22) are investigated by atomic force microscopy (AFM). It is found that A16-22 peptides quickly aggregate from monomers to oligomers and flake-like structures, and finally to fibrils. In particular, unusual morphology change is observed at the early stage of aggregation, that is, the originally formed flake-like structures would disappear in the followed aggregation processes. To figure out the evolution of the flake-like structures, in-situ AFM imaging is carried out in liquid to reveal the real-time morphology changing of A The results provide clear evidences that the flake-like structures are in unstable intermediate state, which would be dissolved into oligomers or short protofibrils for reorganization. Further experiments of Thioflavin T (ThT) fluorescence and attenuated total reflectance Fourier transform infrared (ATR-FTIR) suggest that those flake-like structures contain -sheet components.
Results
Figure 1 Ex situ AFM images of Aβ after incubating for four different time periods: (a) 0.5 , (b) 1.0 , (c) 5 , and (d) 24 hour. The zoomed image of a typical flake is given in the inset of (b).
Figure 2 In-situ AFM images of Aβ16-22 aggregation in liquid on mica substrate with a concentration of 0.1 mM. (a) - (d): images obtained at 0.5, 1.0, 1.5 and 2.0 hours after preparation, respectively. White box and ellipse show the changing process of the same Aβ16-22 aggregates.
Figure 3 (a): The ThT fluorescence spectra of freshly prepared and incubated Aβ16-22 solutions mixed with ThT solution. The spectrum of control ThT solution is also measured to excluding the influence of laser intensity in different measurements; (b): The fluorescence intensity of ThT bound to A16–22 as a function of incubation time. The change at the early stage within 2 hours is zoomed in (c).
Figure 4 Statistical analyses of flakes. (a): The number of flakes per 1 m2 as a function of incubation time; (b): The height (blue) and length (black) of flakes as a function of incubation time, together with the linear fit for height and the exponential fit for length.
Conclusion
In the present study, we have investigated the full aggregation process of Aβ16-22, especially at its early aggregation stages, with in-situ and ex-situ AFM imaging. Our results show that during the aggregation process of Aβ16-22, unstable flakes are observed after half an hour’s incubation and these flakes would be dissolved afterwards while some of them would transfer to protofibrils. Associated with the significance of conformational transformation, secondary structure information is achieved by ThT fluorescence and ATR-FTIR. The results suggest that the flakes are in unstable -sheet structures.
Figure 5 ATR-FTIR spectra of Aβ The absorbance curves of the samples with the incubation time of 15, 30, 60, and 100 minutes are displayed. The intensities of the two peaks at 1625 and 1685 cm-1 are plotted as a function of incubation time in the inset.
Figure 6 Schematic diagram illustrates a possible aggregation
process of Aβ16-22 in neutral pH.
D Lin, Y Luo, S Wu, Q Ma, G Wei, X Yang, Langmuir, 2014 (11), 3170–3175