Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-␤ structure. However, there is a lack of information relating the 4.8 Å ␤-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-␤ ribbon is composed of relatively flat ␤-sheets rather than being the highly twisted, ␤-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in ␤-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.U nder conditions that destabilize the native state, proteins can self-aggregate into insoluble, fibrillar assemblies (1-3). In the form of amyloid fibrils or fibril precursors, the proteins not only lack their original biological function but also may be harmful to organisms, causing pathologies such as Alzheimer's and prion diseases. Although amyloid precursor proteins do not share any sequence or structural homology, amyloid fibrils are typically unbranched, protease-resistant filaments approximately 100 Å in diameter and composed of Ϸ20-35 Å wide protofilaments, which are sometimes arranged around an electron lucent core (4, 5). Recently, several nonpathological proteins and short peptides have been shown to self-assemble into amyloid-like fibrils (6-9), leading to the suggestion that amyloid formation is a generic property of polypeptide chains (3, 10).The overall morphology of amyloid aggregates depends on the conditions in which fibrillogenesis takes place, and different fibril morphologies are often observed in the same preparation (7,(11)(12)(13)(14). Variable structures also are seen in ex vivo fibrils extracted from amyloidotic tissue (4, 15). The morphological variation seems to be caused by fibrils with a variable number and arrangement of protofilaments. X-ray fiber diffraction studies reveal a characteristic cross-␤ structure with ␤-strands of the precursor protein arranged perpendicular to, and ribbon-like ␤-sheets parallel to, the fibril axis (2, 16, 17). The ␤-strand repeat has also been directly visualized by cryo-EM (8). However, there is a lack of three-dimensional (3D) structural information on how the 4.8 Å ␤-strand repeat relates to the overall fibril assembly.The polypeptide hormone insulin has a mainly helical native structure, with its two polypeptide chains linked by two interchain and one intra-chain disulfide bonds (18). In vitro, insulin is readily converted to an inactiv...

Fourier transform infrared spectroscopy~FTIR!, circular dichroism~CD!, and electron microscopy~EM! have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like a-helical structure. On heating to 70 8C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from a-helical to b-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ;450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.Keywords: CD; electron microscopy; fibril formation; FTIR; insulin; X-ray diffractionThe amyloid diseases, which include Alzheimer's disease, the spongiform encephalopathies and type II diabetes, are characterized by the abnormal self-assembly and deposition of proteinaceous material into insoluble ordered

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

No abstract