The protofilament structure of insulin amyloid fibrils

Abstract

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...

Kinetic theory of amyloid fibril templating

“…28,29 The second timescale is t bond ( ), which is the average lifetime of a molecule attached to the fibril end before thermal fluctuations sever the bonds allowing it to diffuse away. This lifetime is a function of , the strength of the bonds holding the molecule onto the fibril end.…”

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies

“…The determination of detailed structures of the mature fibrils is also challenging due to their intractable and frequently heterogeneous nature, which again seriously limits the application of the traditional methods of structural biology such as solution NMR spectroscopy and X-ray diffraction techniques. Structural information concerning amyloid fibrils has, however, been obtained from atomic force microscopy (AFM) [30,31], FTIR [32,33], X-ray fibre diffraction studies [17], cryoelectron microscopy [34,35], hydrogen/deuterium exchange analysed by mass spectrometry and NMR [36][37][38][39] and solid state NMR [40,41]. Important information about both the structures of the aggregates and the mechanism of their formation has also been obtained by using methods such as limited proteolysis [42,43], systematic site-directed mutagenesis [44][45][46], and the analysis of the effects of interactions with specific antibodies.…”

Application Potential of Food Protein Modification

“…Near the isoelectric pH (Arnaudov & de Vries, 2005), or at high salt concentration (Arnaudov & de Vries, 2006;Veerman et al 2002), spherical or amorphous aggregates are formed. That aggregate morphologies cannot always be categorized as either fibrillar or amorphous in a clear-cut manner was shown by various groups observing substantial heterogeneity in aggregate morphology, also called polymorphism, within the same preparation (Bauer et al, 1995;Jiménez et al, 2002Jiménez et al, , 1999. As a consequence, gels formed under these conditions of -lactoglobulin are particulate and the particle size depends on heating temperature and heating rate (Bromley et al, 2006).…”