The early stages of amyloid formation: biophysical and structural characterization of human calcitonin pre-fibrillar assemblies.
Amyloid fibril formation is a nucleation dependent process characterized by a lag-phase prior to the appearance of detectable amyloid fibrils. While the three-dimensional structure of amyloid fibrils at atomic resolution is just beginning to be elucidated, the early process of monomers assembly into oligomers is less understood. Understanding the dynamic processes that lead to the formation of these intermediates is highly important as these assemblies might be the most pathological ones. Here, we investigated the biophysical and structural features characterizing the early stage assemblies formed by the human hormone calcitonin. We calculated the initial nucleus size by experimentally determining the dependence between the lag-time length and the hCT concentrations. We used size exclusion chromatography and dynamic light scattering in order to characterize the dynamic growth process of preliminary intermediates transformed into larger structures. The early structures were visualized using high-resolution transmission electron microscopy. Annular pore-like structures were observed along with protofibrilar structures. This observed morphology is similar to structures revealed during the fibrillization processes of beta-amyloid, alpha-synuclein, and islet amyloid polypeptide, suggesting that these intermediates represent a generic early structure conformation. The results introduced here imply that a variety of intermediate assemblies are formed during the early stages of amyloid fibril formation. The characterizing of their structural features and assembly kinetics will contribute to the rational design of inhibitors directed towards early structure assemblies.
Avidan-Shpalter
Carmit
C
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, 69978, Israel.
Gazit
Ehud
E
D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D052160:Benzothiazoles; D002116:Calcitonin / Q000737:chemistry*; D002850:Chromatography, Gel; D005456:Fluorescent Dyes; D006801:Humans; D008027:Light; D046529:Microscopy, Electron, Transmission; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013844:Thiazoles / Q000737:chemistry
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T; D002116:Calcitonin