Formation of Amyloid Fibrils via Longitudinal Growth of Oligomers

Abstract

Mature amyloid fibrils are believed to be formed by the lateral association of discrete structural units designated as protofibrils, but this lateral association of protofibrils has never been directly observed. We have recently characterized a thioesterase from Alcaligenes faecalis, which was shown to exist as homomeric oligomers with an average diameter of 21.6 nm consisting of 22 kDa subunits in predominantly beta-sheet structure. In this study, we have shown that upon incubation in a 75% ethanol solution, the oligomeric particles of protein were transformed into amyloid-like fibrils. TEM pictures obtained at various stages during fibril growth helped us to understand to a certain extent the early events in the fibrillization process. When incubated in 75% ethanol, oligomeric particles of protein grew to approximately 35-40 nm in diameter before fusion. Fusion of two oligomers of 35-40 nm resulted in the formation of a fibril. Fibril formation was accompanied by a reduction in the diameter of the particle to approximately 20-25 nm along with concomitant elongation to approximately 110 nm, indicating reorganization and strengthening of the structure. The elongation process continued by sequential addition of oligomeric units to give fibers 500-1000 nm in length with a further reduction in diameter to 17-20 nm. Further elongation resulted in the formation of fibers that were more than 4000 nm in length; the diameter, however, remained constant at 17-20 nm. These data clearly show that the mature fibrils have assembled via longitudinal growth of oligomers and not via lateral association of protofibrils.

Site-Specific Blockade of RAGE-VdPrevents Amyloid-β Oligomer Neurotoxicity

“…1B, A␤Os) (Losic et al, 2006;Mastrangelo et al, 2006;Moore et al, 2007). These observations conform to previous studies indicating that mature amyloid fibrils occur through a number of intermediate structural forms referred to as oligomers (Arimon et al, 2005;Shahi et al, 2007). Although the precise oligomer stoichiometry remains unclear, the preparations were predominantly free of protofibrils and entirely free of fibrils as indicated by the absence of significant variation in Congo red binding during this period of time (Fig.…”

Principles, approaches, and challenges for predicting protein aggregation rates and shelf life

“…Using this nomenclature, the osmotic second virial coefficient (B (16) In practice, Dg ðaÞ 12 ðrÞ is not accessible directly via experiment; the exception is at high protein concentrations ($10 2 g/L) using specialized techniques based on X-ray and neutron scattering. 113,114 Therefore, one often must assume a physically reasonable form for Dg ðaÞ 12 ðrÞ and approximate or fit model parameters to best describe the experimental system of interest.…”

“…8,12,13 By default, nonfibrillar high-MW aggregates are often termed amorphous. 10,[14][15][16] There is recent evidence that amorphous, high-MW aggregates for pharmaceutically relevant systems such as G-CSF are well-described as linear, semi-flexible polymers. 10 An empirically common feature among aggregates from a variety of pharmaceutical systems is that the constituent monomers often show measurably elevated b-sheet 28 structure when compared to the folded or unfolded parent monomers.…”

“…Based on these considerations and Eqs. (14)-(16), the folded-state quantities therefore serve as rigorously based, albeit surrogate, parameters to inform indirectly on k obs .Figure 2and the calculations described below and in Supporting Information show qualitative and quantitative relationship between osmotic virial coefficients and equilibrium constants for native and non-native dimer formation. For simplicity and illustration purposes, Dg ðaÞ 12 ðrÞ is based on a colloidal interaction model typical of those used to model liquid-phase protein-protein interactions and phase behavior 20,112,[115][116][117][118].…”

The use of mass spectrometry to study amyloid‐β peptides

“…In structural terms, the process by which Ab assembles from monomers into oligomers and fibrils is not well understood, but it is generally agreed that the oligomers represent an intermediate stage of the processes that lead to fibril formation, as schematized in Figure 2 (Lomakin et al, 1997;Montalto, Farrar, & Tan Hehir, 2007;Shahi et al, 2007;Zhang et al, 2009). …”