Determination of size of folding nuclei of fibrils formed from recombinant Aβ(1-40) peptide

Abstract

We have developed a highly efficient method for purification of the recombinant product Aβ(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aβ(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one "monomer" and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.

Rosetta Stone for Amyloid Fibrils: The Key Role of Ring-Like Oligomers in Amyloidogenesis

“…Models that could account for the low-angle meridional reflections in A␤ peptides 9-28 and 1-40 can be: (a) periodic arrangement of discrete objects along the fibril axis; (b) staggered arrangement of subfibrils; and (c) twisting of the fibril. Case (a) supports our model both for A␤ and A␤ [37,38,43] and for the short peptides of 10 residues long [40] having a ladder-type arrangement of oligomers (see Figs. 4 and 5).…”

“…A number of models has been proposed for the process of assembling of amyloid fibrils, in which the interaction of oligomer particles in different ways leads to formation of elongated polymers of various amyloid proteins [18,[25][26][27][28][29]. Our own studies of different amyloidogenic proteins (insulin, A␤ peptides, and their fragments, amyloidogenic fragments of Bgl2 protein) show that at the beginning of fibril formation a large number of oligomer ring-like structures is observed as well as a small number of short thin fibrils with a diameter comparable to that of oligomers [36][37][38][39][40]. As the time of incubation increases, the fraction of oligomer particles decreases, fibrils grow to several micrometers in length, and fibrils of different morphologies are formed.…”

“…In the cross section of a fibril, such packing is seen as a ring structure, the internal diameter of the ring being about 15-20Å [70]. Surprisingly, it is possible to see exactly the same morphology of A␤ peptide fibrils using the EM method [36][37][38]44]. However, the knowledge accumulated during the recent years and the remaining unsolved problems of the structure organization as well as our data on amyloid peptides and their fragments allow us to propose another view on the process of fibrillogenesis, and the structural organization of amyloid fibrils.…”

Apolipoprotein E impairs amyloid‐β fibril elongation and maturation

“…The selective binding to oligomeric Aβ assemblies acting as nuclei is likely the reason to the strong effect seen also at a 1 : 1500 substoichiometric ratio between ApoE and Aβ. In this context, the size of the Aβ nuclei is also of interest to discuss and it has previously been shown that the minimal size of an Aβ nuclei is only around 2–3 assembled peptides . It is from these results not possible to elucidate whether ApoE can interact already at this early point or if the formation of larger prefibrillar structures first is required.…”

Determination of amyloid core regions of insulin analogues fibrils

“…Amyloids are characterized by high resistance to chemical and physical influences, such as changes in pH, temperature, pressure, and protease treatment [36][37][38][39][40]. The latter property can be used in mass spectrometry analysis of amyloidogenic regions in insulin fibrils and its analogues using the method developed previously for this purpose [41][42][43]. Targeted action to the identified amyloidogenic sequences can significantly slow down or prevent amyloidogenesis [44].…”