Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation

Abstract

Recent experimental evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogendeuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addition to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further analysis using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur. aggregation ͉ HD exchange ͉ misfolding intermediates ͉ PI3-SH3

Structural Properties of Pore-Forming Oligomers of α-Synuclein

“…36 Incontrast,pulse-labelinghydrogen-deuterium exchange coupled to mass spectrometry indicated that aggregation of an SH3 domain occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. 37 The solidstate NMR data shown in Figure 7 are in agreement with a nonfibrillar -structure of soluble oligomers of RS. Moreover, the observation that the soluble RS oligomers but not the fibrils formed pores in lipid bilayers points to a lower importance of -structure for pathological function.…”

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

“…The amyloid-forming properties of the SH3 domain of bovine phosphatidylinositol-3-kinase (PI3-SH3), an 86-residue protein, have been interrogated in vitro [12,73]. Under acidic pH conditions, P13-SH3 adopts a compact denatured state which slowly forms a gel that consists of typical amyloid fibrils [74].…”

“…First, the protein monomer may well be present in a co-populated mixture of folded, partially folded, and highly unfolded families of conformers, any one (or more) of which may be capable of initiating the assembly process [11]. Next, although early reports indicated that the amyloid fibrils were the primary pathogenic agent, recent evidence has suggested that smaller aggregates populated during fibril assembly are more likely to be responsible for pathogenesis [12][13][14][15]; for example, it has been suggested that A␤ oligomers could be the cause, rather than a product of, Alzheimer's disease [16]. This suggestion makes the characterization of all species present within the heterogeneous ensembles that exist during the aggregation process both necessary and highly important.…”

Crucial role of nonspecific interactions in amyloid nucleation

“…Indeed, there is strong evidence that the disordered oligomers rather than fully grown fibrils are the main pathogenic species in protein aggregation diseases (31-33). As such, defining the role of the prefibrillar oligomers during amyloid formation will be crucial to develop intervention strategies that target these species (1,30,34,35).Mutations in the polypeptide sequence and extrinsic changes in the experimental conditions are known to alter the concentrations of aggregated species, their size, and their cytotoxicity (25,(36)(37)(38)(39). For instance, mutations that increase hydrophobicity of the Alzheimer's β-peptide (Aβ1-42) have a pronounced effect on its aggregation behavior and the size distribution of the resulting oligomers (23)(24)(25)(26)40), promoting toxicity and expediting the fibrillization process.…”

“…Mutations in the polypeptide sequence and extrinsic changes in the experimental conditions are known to alter the concentrations of aggregated species, their size, and their cytotoxicity (25,(36)(37)(38)(39). For instance, mutations that increase hydrophobicity of the Alzheimer's β-peptide (Aβ1-42) have a pronounced effect on its aggregation behavior and the size distribution of the resulting oligomers (23)(24)(25)(26)40), promoting toxicity and expediting the fibrillization process.…”