Evidence suggests that oligomers of the 42-residue form of the amyloid β-protein (Aβ), Aβ42 play a critical role in the etiology of Alzheimer’s disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aβ42 that occur at the earliest stages of aggregation. We observe features that can be attributed to monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aβ42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 μM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear pre-protofibrillar β-sheet structures. The pre-protofibrils are a single Aβ42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the pre-protofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aβ40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures.

Abstract. Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobilitybased mass spectrometry experiments, molecular dynamics simulations, and highresolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of interpeptide hydrogen bonding.

The aggregation of human islet amyloid polypeptide (hIAPP) has been closely associated with the pathogeny of type 2 diabetes mellitus (T2DM) and destruction of pancreatic islet β-cells. Several amyloidogenic domains within the hIAPP sequence capable of self-association have been identified. Among them is the 8-20 region of hIAPP, which has formed β-sheet fibrils despite being contained within an α-helical region of full-length hIAPP. To further understand the propensity of this region for self-assembly, two peptide fragments were compared, one consisting of the residues 8-20 (WT) and a mutant fragment with a His18Pro substitution (H18P). The conformational distribution and aggregation propensity of these peptides was determined using a combination of ion mobility mass spectrometry and atomic force microscopy. Our results reveal that the two peptide fragments have vastly differing assembly pathways. WT produces a wide range of oligomers up to decamer whereas the H18P mutant produces only low order oligomers. This study confirms the propensity of the 8-20 region to aggregate from its native α-helical structure into amyloid β-sheet oligomers and highlights the significance of the charged His18 in the aggregation process.

Protein aggregation is typically attributed to the association of homologous amino acid sequences between monomers of the same protein. Coaggregation of heterogeneous peptide species can occur, however, and is implicated in the proliferation of seemingly unrelated protein diseases in the body. The prion protein fragment (PrP) and human islet amyloid polypeptide (hIAPP) serve as an interesting model of nonhomologous protein assembly as they coaggregate, despite a lack of sequence homology. We have applied ion-mobility mass spectrometry, atomic force microscopy, circular dichroism, and high-level molecular modeling to elucidate this important assembly process. We found that the prion fragment not only forms pervasive hetero-oligomeric aggregates with hIAPP but also promotes the transition of hIAPP into its amyloidogenic β-hairpin conformation. Further, when PrP was combined with non-amyloidogenic rIAPP, the two formed nearly identical hetero-oligomers to those seen with hIAPP, despite rIAPP containing β-sheet breaking proline substitutions. Additionally, while rIAPP does not natively form the amyloidogenic β-hairpin structure, it did so in the presence of PrP and underwent a conformational transition to β-sheet in solution. We also find that PrP forms hetero-oligomers with the IAPP fragment but not with the "aggregation hot spot" IAPP fragment. PrP apparently induces IAPP into a β-hairpin structure within the PrP:IAPP heterodimer complex and then, through ligand exchange, catalytically creates the amyloidogenic β-hairpin dimer of IAPP in significantly greater abundance than IAPP does on its own. This is a new mechanistic model that provides a critical foundation for the detailed study of hetero-oligomerization and prion-like proliferation in amyloid systems.

Type-2 diabetes mellitus (T2DM) is a disease hallmarked by improper homeostasis within the islets of Langerhans of the pancreas. The most critical species affected is insulin, which is produced by the β-cells of the islets, but there are a number of other species copackaged and cosecreted within the insulin granules. This includes zinc, which exists in high (millimolar) concentrations within the β-cells, and islet amyloid polypeptide (IAPP), which is an amyloid peptide thought to induce β-cell apoptosis through self-association into toxic amyloid oligomers. Zinc is essential in the packaging of crystalline insulin within the vesicles but it can also bind and interact with IAPP. This implies a complex relationship between all three species and diabetes, particularly in the structure and function of toxic IAPP aggregates. Atypical (low or high) concentrations of zinc generally appear to correlate with increased hIAPP aggregation, whereas physiological zinc concentrations have an inhibitory effect. To better understand how zinc ions alter the monomer and oligomer structure of hIAPP in vitro, we employ a combination of ion mobility mass spectrometry and atomic force microscopy. We observe an increase in the extended β-hairpin conformation of hIAPP when it is bound to zinc. With sufficiently low concentrations of zinc this could result in an association site for zinc-free hIAPP, promoting amyloid aggregation. At high zinc concentrations, we see the appearance of a secondary zinc association site whose coordination could account for the loss of inhibition at high zinc concentrations. Generally, it appears that zinc preferentially stabilizes the β-hairpin conformation of hIAPP and the population of zinc-bound hIAPP in solution determines what effect this has on amyloid aggregation.