Direct Observation of Amyloid Fibril Growth Monitored by Thioflavin T Fluorescence

Abstract

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of ␤2-microgobulin (␤2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent ␤2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid ␤-peptide.There is an increasing body of evidence showing that many proteins including the Alzheimer's amyloid ␤-peptide (A␤), 1 prion protein, transthyretin, and ␤2-microgobulin (␤2-m) tend to misfold and aggregate into amyloid fibrils (1-3). Moreover, several proteins not known to be involved in disease and various polyamino acids have been shown to form amyloid fibrils in vitro under carefully selected conditions (4, 5). Although no sequence or structural similarity between the amyloid precursor proteins has been found, amyloid fibrils share several common structural and spectroscopic properties (6). Irrespective of the protein species, electron microscopy and x-ray fiber diffraction indicate that the amyloid fibrils have relatively rigid structures with diameters of 10 -15 nm consisting of cross-␤-strands. Making use of an NMR technique in combination with hydrogen/deuterium exchange of amide protons and dissolution of amyloid fibrils by dimethyl sulfoxide, we have shown that the amyloid fibrils of ␤2-m are stabilized by a hydrogenbond network which is more extensive than that in the native state (7).Amyloid fibril formation is considered to be a nucleation-dependent process in which non-native precursor proteins slowly associate to form the nuclei (8). This process is followed by an extension reaction, where the nucleus grows by sequential incorporation of more precursor protein molecules. This model has been validated by the observation that fibril extension kinetics is accelerated by the addition of preformed fibrils, i.e. by a seeding effect. However, the mechanism of fibril formation by individual polypeptide chains is not completely understood, and there are several variations of the nucleation-dependent model (9, 10). To address the mechanism of amyloid fibril formation, it is important to observe the process at the singlefibril level. Recently, epifluorescence with a newly introduced fluorescent dye (11) and atomic force microscopy (AFM) (12, 13) have been utilized for the d...

Amyloid-β peptide binds with heme to form a peroxidase: Relationship to the cytopathologies of Alzheimer’s disease

“…Aggregates of A␤ were prepared by allowing spontaneous aggregation in an A␤ solution at 25°C for at least 24 h, which was then divided into aliquots and stored at Ϸ20°C. The aggregation of A␤ was measured by assaying the fluorescence of TfT, which increases upon binding to A␤ aggregates [excitation filter of 435 nm (bandwidth, 5 nm) and emission filter of 486 nm (bandwidth, 10 nm)] (46,47).…”

Stepwise dynamics of epitaxially growing single amyloid fibrils

“…The kinetics of amyloid assembly are typically investigated in bulk assays that often hide details of the process. Recently, the growth of individual amyloid fibrils was observed by using atomic force microscopy (AFM) (6, 7) and fluorescence microscopic methods (8,9). In some experiments, a stop-and-run phenomenon was observed, during which the fibril growth was temporarily halted (9,10), whereas others pointed to a constant growth rate (8,9).…”

Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet

“…However, obtaining structural information about intermediate species is difficult due to their transient nature. Solid-state NMR (ssNMR) and X-ray crystallography provide high-resolution structures of fibrils (4,5) and optical techniques can track structural changes in real time (6,7), but few techniques have both the structural and the temporal resolution to extract specific structural details about intermediates. Fragments have been trapped in intriguing oligomeric structures that may represent intermediate states (5,8) and transient secondary structures are known to exist from circular dichroism measurements and other experiments (9)(10)(11), but for full-length proteins it has been difficult to identify the specific residues that contribute to the secondary structure and thus understand their role in the aggregation mechanism.…”