Understanding amyloid aggregation by statistical analysis of atomic force microscopy images

Abstract

The aggregation of proteins is central to many aspects of daily life, including food processing, blood coagulation, eye cataract formation disease and prion-related neurodegenerative infections [1][2][3][4][5] . However, the physical mechanisms responsible for amyloidosis-the irreversible fibril formation of various proteins that is linked to disorders such as Alzheimer's, Creutzfeldt-Jakob and Huntington's diseases-have not yet been fully elucidated [6][7][8][9] . Here, we show that different stages of amyloid aggregation can be examined by performing a statistical polymer physics analysis of single-molecule atomic force microscopy images of heat-denatured b-lactoglobulin fibrils. The atomic force microscopy analysis, supported by theoretical arguments, reveals that the fibrils have a multistranded helical shape with twisted ribbon-like structures. Our results also indicate a possible general model for amyloid fibril assembly and illustrate the potential of this approach for investigating fibrillar systems.Protein self-assembly is a wide-ranging phenomenon and is of great importance in several areas of science. The reversible formation of fibrils from globular proteins is a phenomenon occurring naturally, in vivo, for proteins such as actin and tubulin 10,11 . Other well-known examples of protein fibrillation include the irreversible amyloid fibril formation of various proteins implicated in neurological disorders such as Alzheimer's, Creutzfeldt-Jakob or Huntington's diseases. Typically, these fibrils have long, unbranched, and often twisted structures that are a few nanometres in diameter 1,8 . However, many peptides and proteins, including many globular food proteins that are used as gelling agents, foaming agents or emulsifiers, can also form amyloid-like structures in vitro. Such structures can possess desirable mechanical properties and can be used to create useful textures and structures 12,13 .An example of a fibril formation of globular proteins is provided by the fine-stranded heat-set gels formed by heating solutions of various globular food proteins such as ovalbumin, bovine serum albumin 12 and b-lactoglobulin 13 . b-Lactoglobulin has been particularly well studied, because it represents both a relevant model system and a major whey protein of interest to the food industry [14][15][16][17][18][19][20][21][22] .Despite the fact that the individual parameters controlling the aggregation of globular proteins into amyloid fibrils have been well identified, the driving force for such an aggregation process still remains obscure. 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...

Non-equilibrium nature of two-dimensional isotropic and nematic coexistence in amyloid fibrils at liquid interfaces

“…This results in an increase in R 2 2D , and hence a twofold rise in rigidity from l b ¼ 1.5 to 3.0 mm. The l b ¼ 1.5 mm is in excellent agreement with previously reported l b values of infinitely diluted b-lactoglobulin fibrils 41 , showing that interactions between fibrils are still negligible at the lowest density considered here (see Supplementary Fig. S3 for corresponding particle tracking data).…”

Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method

“…This approach has been successfully employed to determine the Young's modulus of b-lactoglobulin fibrils, for which the packing schemes of the protofilaments are now well-established. 15 A first success of the PF-QNM method has been to show that the measured values of Young's modulus and those extracted by this indirect procedure were in excellent agreement. 22 This indirectly points to another very important issue in amyloid fibrils, that is, different polymorphic amyloid forms of the same protein or peptide should possess different area moments of inertia, different persistence lengths, but identical Young's moduli.…”

Coexistence of ribbon and helical fibrils originating from hIAPP 20–29 revealed by quantitative nanomechanical atomic force microscopy

“…2A) reveals the coexistence of two types of hIAPP [20][21][22][23][24][25][26][27][28][29] nanostructures, namely, the ribbon and the twisted fibrils. The twisted fibrils are considered to be the mature species of the amyloid self-assembly due to their structural complexity (11,36). However, three kinds of twisted fibrils with different diameters, as indicated by the terms fibril 1, fibril 2, and fibril 3, are depicted in Fig.…”

“…Atomic force microscopy (AFM) is capable of obtaining nanoscale resolution of individual molecules or supermolecular structures. This method also allows for the analysis of the selfassembly mechanism and the driving force of aggregation (10)(11)(12)(13)(14). Importantly, AFM, furthermore, provides the possibility to follow the dynamics to obtain a detailed picture of the amyloid assembly process (15).…”

“…High-resolution solidstate NMR structures of hIAPP [20][21][22][23][24][25][26][27][28][29] fibrils have provided detailed insight into the antiparallel hetero zipper with a twist along the fibril axis (21)(22)(23). However, no uniform detailed theory of the self-assembly behavior is available (11) and, in particular, the transitions between different intermediates during fibrillation remain to be elucidated.…”