Early aggregation preceding the nucleation of insulin amyloid fibrils as monitored by small angle X-ray scattering

Abstract

The nucleation event of amyloid fibrils is one of the most crucial processes that dictate the timing and rate of the pathology of diseases; however, information regarding how protein molecules associate to produce fibril nuclei is currently limited. In order to explore this issue in more detail, we performed time-resolved small angle X-ray scattering (SAXS) measurements on insulin fibrillation, in combination with additional multidirectional analyses of thioflavin T fluorescence, FTIR spectroscopy, light scattering, and light transmittance, during the fibrillation process of bovine insulin. SAXS monitoring revealed that insulin molecules associated into rod-like prefibrillar aggregates in the very early stage of the reaction. After the formation of these early aggregates, they appeared to further coalesce mutually to form larger clusters, and the SAXS profiles subsequently showed the further time evolution of conformational development towards mature amyloid fibrils. Distinct types of structural units in terms of shape in a nano-scale order, cross-β content, and thioflavin T fluorescence intensity were observed in a manner that was dependent on the fibrillation pathways. These results suggest the presence of diverse substructures that characterize various fibrillation pathways, and eventually, manifest polymorphisms in mature amyloid fibrils.

A specific form of prefibrillar aggregates that functions as a precursor of amyloid nucleation

“…Furthermore, several techniques including ion mobility-mass spectrometry 10 , ESR 12 , time-lapse solid-state NMR 11 , and the development of methodology using hybrid peptides 33 or a latent fluorophore 34 recently revealed the successful conformational conversion of aggregates to β-sheet-rich fibrils. Previous finding also showed that aggregates convert to mature amyloid fibrils in parallel with their mutual association 35 , which may also be categorized as the NCC mechanism in a broad context.…”

Recent progress on understanding the mechanisms of amyloid nucleation

“…This figure also represents that, in parallel with the typical templated assembly of monomers (top right panel), the oligomer species undergoes conformational conversion upon association with preformed nuclei in the step of elongation, a discussion of which is beyond the scope of this review were further investigated by time-resolved SAXS measurements combined with thioflavin T fluorescence, Fouriertransform infrared and light scattering studies. It was revealed that insulin molecules associated into rod-like prefibrillar intermediates in the very early time range of the reaction, and that after their further coalescing to form larger clusters, a conformational conversion towards mature amyloid fibrils occurred (Chatani et al 2015) (Fig. 4).…”

“…In the case of the two-step nucleation mechanism of crystals, the feature of prenucleation clusters is often depicted as highly condensed and liquid-like particles of solutes, inside of which the formation of crystalline nucleus would be facilitated by increasing chances of interatomic or intermolecular interactions (Erdemir et al 2009;Vekilov and Vorontsova 2014). Chatani et al (2014Chatani et al ( , 2015. In this reaction, rod-like shaped prefibrillar intermediates having a partial β-sheet structure (gray regions) are initially formed.…”

Dynamic Measurements of Bovine Serum Albumin Aggregation Using Small Angle Neutron Scattering

“…Small-angle neutron scattering (SANS) has been successfully used to analyse static protein aggregate samples [28][29][30], but has been somewhat overlooked by general reviews of techniques for analysing protein aggregation. Particular advantages include probing structures over a range of length scales and minimal beam damage to samples.…”