The assembly mechanisms of amyloid fibrils, tissue deposits in a variety of degenerative diseases, is poorly understood. With a simply modified application of the atomic force microscope, we monitored the growth, on mica surface, of individual fibrils of the amyloid ␤25-35 peptide with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid (up to 300-nm ؊1 ) growth phases that extended the fibril by Ϸ7 nm or its integer multiples were interrupted with pauses. Stepwise dynamics were also observed for amyloid ␤1-42 fibrils growing on graphite, suggesting that the discontinuous assembly mechanisms may be a general feature of epitaxial amyloid growth. Amyloid assembly may thus involve fluctuation between a fast-growing and a blocked state in which the fibril is kinetically trapped because of intrinsic structural features. The used scanning-force kymography method may be adapted to analyze the assembly dynamics of a wide range of linear biopolymers.atomic force microscopy ͉ beta-amyloid ͉ growth dynamics ͉ self-assembly

Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid ␤ (A␤) peptides 1-40 or 25-35, ␤-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for A␤1-40 and A␤25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the ␤-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of ␤-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.Amyloid fibrils are self-associating filamentous structures formed from the 39 -43-residue-long amyloid ␤-peptide (A␤) 1 or its subfragments (1). The deposition of amyloid oligomers (2) and fibrils is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease (3) and other disorders (4). The structure of A␤-fibrils has for long been enigmatic because of insoluble aggregate formation that precludes the use of standard structural methods such as x-ray crystallography and solution NMR. Recent data from site-directed spin labeling (5), and particularly from solid-state NMR experiments (6, 7), have formed the basis of a high resolution model of the A␤1-40 fibril: ␤-hairpins lying perpendicular to the fibril axis are associated into ␤-sheets that line up to form protofilaments, which are then assembled parallel into fibrils. Protofilaments are thus thought to represent an ϳ2-3-nm-diameter structural unit within the amyloid fibril (1). During amyloidogenesis the formation of fibrils is preceded by the appearance of globular aggregates that are thought to fuse, by not fully understood mechanisms, into fibrillar structures (8). Recently, curved, beaded, ϳ200-nm-long and ϳ6 -8-nm-wide fibrillar precursors were described to appear in the amyloidogenetic pathway, which were called protofibrils (9 -12). The protofibrils are thought to go through a structural transition on their way to forming the amyloid fibril. The exact nature of structural dynamics within the amyloid fibril related to amyloidogenesis, however, remains to be resolved.Single molecule manipulation experiments have in the recent past provided unique and unprecedented insights not only into the structure and elasticity but also into mechanically driven transitions of molecular systems (13)(14)(15)(16)(17)(18)(19)(20)(21). In the present work we mechanically manipulated amyloid fibrils formed from either A␤1-40 or A␤25-35 peptides. We showed that filamentous entities most likely corresponding to ␤-sheets can be "unzipp...

Nature provides numerous examples of self-assembly that can potentially be implemented for materials applications. Considerable attention has been given to one-dimensional cross-β or amyloid structures that can serve as templates for wire growth or strengthen materials such as glue or cement. Here, we demonstrate controlled amyloid self-assembly based on modifications of β-solenoid proteins. They occur naturally in several contexts (e.g., antifreeze proteins, drug resistance proteins) but do not aggregate in vivo due to capping structures or distortions at their ends. Removal of these capping structures and regularization of the ends of the spruce budworm and rye grass antifreeze proteins yield micron length amyloid fibrils with predictable heights, which can be a platform for biomaterial-based self-assembly. The design process, including all-atom molecular dynamics simulations, purification, and self-assembly procedures are described. Fibril formation with the predicted characteristics is supported by evidence from thioflavin-T fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy. Additionally, we find evidence for lateral assembly of the modified spruce budworm antifreeze fibrils with sufficient incubation time. The kinetics of polymerization are consistent with those for other amyloid formation reactions and are relatively fast due to the preformed nature of the polymerization nucleus.

No abstract

BackgroundDefects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated.Methodology/Principal FindingsWe investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway.Conclusions/SignificanceOur observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.