Abstract
Oligomers which form during amyloid fibril assembly are considered to be key contributors towards amyloid disease. However, understanding how such intermediates form, their structure, and mechanisms of toxicity presents significant challenges due to their transient and heterogeneous nature. Here, we discuss two different strategies for addressing these challenges: use of (1) methods capable of detecting lowly-populated species within complex mixtures, such as NMR, single particle methods (including fluorescence and force spectroscopy), and mass spectrometry; and (2) chemical and biological tools to bias the amyloid energy landscape towards specific oligomeric states. While the former methods are well suited to following the kinetics of amyloid assembly and obtaining low-resolution structural information, the latter are capable of producing oligomer samples for high-resolution structural studies and inferring structure-toxicity relationships. Together, these different approaches should enable a clearer picture to be gained of the nature and role of oligomeric intermediates in amyloid formation and disease.
Highlights
Amyloid fibril formation is a complex and multifaceted process, characterized by the self-assembly of proteins or peptides into insoluble cross-β deposits [1,2] (Fig. 1A)
ESI-MS can be directly coupled to other methods, such as ion mobility spectrometry (IMS; which allows oligomers with the same m/z ratio to be separated based on size and shape) [41,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133], hydrogen-deuterium exchange and related covalent labelling experi ments [134,135,136,137,138,139,140,141,142,143,144,145,146,147], and infrared spec troscopy (IR) [39,40] to yield structural information about the different species present in an aggregating mixture
A11 was raised against an oligomeric mimic of Aβ40, this polyclonal antibody recognizes pre-fibrillar, toxic oligomers formed by a range of amyloid proteins with diverse primary sequences and native folds, suggesting that toxicity is associated with a common set of structural features [6]
Summary
Amyloid fibril formation is a complex and multifaceted process, characterized by the self-assembly of proteins or peptides into insoluble cross-β deposits [1,2] (Fig. 1A). We discuss approaches through which observations can be made without intentional pertur bation of the self-assembly process (e.g. using solution NMR, single particle methods, and native ESI-MS), as well as those through which the self-assembly equilibrium is deliberately modified to gain information about specific species These “non-perturbing” and “perturbing” methods are complementary: most non-perturbing techniques are best suited to the determination of kinetic parameters (yielding information about rates of interconversion and population lifetimes), while the res olution of structural information they can obtain is typically lower. By contrast, perturbing methods offer the opportunity to analyze species using high-resolution structural methods, and enable detailed structure-toxicity relationships to be determined The combination of both of these approaches provides an ideal experimental toolkit for generating more complete descriptions of the molecular mechanisms of amyloid fibril formation and their associated cellular toxicity
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