Ultrastructural Organization of Amyloid Fibrils byAtomic Force Microscopy

Abstract

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.

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.…”

“…Tests based on the detection of protease resistant forms of PrP: These tests are based on the observation that the C-terminal region of PrP (usually denoted as PrP [27][28][29][30] ) is resistant to a treatment with the proteinase K in PrP Sc but not in PrP C . Thus, the PrP C species is first eliminated from the sample by a proteolytic treatment.…”

Understanding amyloid aggregation by statistical analysis of atomic force microscopy images

“…Similarities between the formation of b-lactoglobulin fibrils and amyloid protein aggregation have so far only been suggested, but not confirmed 17,19 . Amyloid fibrils obtained by different aggregation mechanisms and from other proteins do show evidence of helical structures formed by twisting of single filaments around each other 26,27 . Our AFM studies of b-lactoglobulin fibrils not only demonstrate analogies between heat-induced protein fibrils and amyloid fibrils, but also clearly reveal similarities in their self-assembly structure and mechanisms, suggesting a possible general model for amyloid fibril assembly.…”

“…Studies devoted to the characterization of fibril structures based on light, neutrons and X-ray scattering methods, being bulk techniques, have only provided an average ensemble picture of the fibrils 23 . Single-molecule techniques such as atomic force microscopy (AFM) and transmission electron microscopy (TEM) have recently emerged to probe amyloid fibrils at the molecular level [24][25][26][27] . Nevertheless, so far, a fully comprehensive picture of the aggregation behaviour among different amyloid fibrils has not yet been achieved.…”

Hyperthin nanochains composed of self-polymerizing protein shackles

“…Fourth, the fibrils studied to date have been relatively thick; for example, the diameters of amyloid and collagen fibrils are 5-20 nm (ref. 12) and 50-500 nm (ref. 13), respectively.…”