Intrinsic Linear Heterogeneity of Amyloid β Protein Fibrils Revealed by Higher Resolution Mass-per-length Determinations

Abstract

Amyloid ␤ proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by darkfield and transmission electron microscopy in a preparation of seeded amyloid ␤ protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when massper-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.Amyloid fibrils are filamentous aggregates formed by many different proteins in various diseases both in and out of the central nervous system (1). Fibrils formed by 40-residue (A␤40) and 42-residue (A␤42) amyloid ␤ (A␤) 2 proteins comprise the amyloid plaques of Alzheimer disease. Considerable effort has been expended to determine how A␤ proteins are folded within fibrils, yet the models emerging from these efforts differ significantly (1-8). Contributing to uncertainty about the internal structure of fibrils is a growing appreciation that A␤ fibrils are polymorphic when examined by transmission EM, cryo-EM, and AFM (7, 9 -19).Two morphological features have particularly important implications with regard to the internal structure of amyloid fibrils. One feature is a periodic narrowing observed in many transmission EM studies suggesting that fibrils twist around their long axis. The other feature is the fibril MPL, measured by dark field or tilted-beam EM. From this measure, the number of filaments comprising the fibril may be determined. We follow the terminology conventions defined by Kodali et al. (12) with the exception that "protofilament" is shortened simply to "filament."Fibrils with different morphologies can be created by varying fibril growth conditions such as temperature, buffer composition, agitation, and protein concentrations. Once formed, fibrils with a given morphology induce monomeric proteins to form "next generation" fibrils with the same morphology (10). Despite attempts to use identical conditions of formation, different laborat...

Intrinsic Structural Heterogeneity and Long-Term Maturation of Amyloid β Peptide Fibrils

“…25 The 2D-IR measurements reported herein now indicate that there are heterogeneities and mobile water molecules within Aβ fibril filaments, and that they lead to an evolution of secondary internal fibril structure over long times. These conclusions are significant to molecular modeling efforts aimed at understanding the formation of amyloid fibrils, and at the design of diagnostic or therapeutic agents that target amyloid fibrils.…”

“…27 It is also unlikely that these fibrils disaggregated and then reaggregated into a different structure over the span of 4 years because the equilibrium monomer concentrations for these samples were measured and found to be undetectable. 25 Chemical changes such as deamidation are possible, and may alter the pathways and kinetics of fibril formation. 37 However, the excitonic structure and morphological appearance of the 4-year-old fibrils in this study are unchanged, and the changes observed are often associated with fast spectral diffusion suggesting that they are due to the presence of mobile water molecules.…”

Distinct Membrane Disruption Pathways Are Induced by 40-Residue β-Amyloid Peptides

“…Whether the peptide aggregates before interacting with the membrane depends on the local A␤ concentration, which can be affected by a variety of factors, such as the efficiency of A␤ elution from the membrane and the clearance of existing A␤ aggregates. In addition, the critical concentration of aggregation for A␤ peptides may also vary in a broad range, from below 0.1 M to above 10 M, as determined previously using a number of techniques (45)(46)(47)(48). Therefore, the interactions between A␤ and the membrane may start from a highly heterogeneous system that involves the co-existence of A␤ monomers and small and large oligomers.…”

Novel Mechanistic Insight into the Molecular Basis of Amyloid Polymorphism and Secondary Nucleation during Amyloid Formation

“…9,10 Previous studies involving in situ atomic force microscopy (AFM) or transmission electron microscopy (TEM) have revealed Aβ42 fibrillogenesis to proceed via the formation of highmolecular-weight protofibrils. 11,12 The kinetics of protofibril formation and stability and the structure of the final fibrils depend greatly upon ambient conditions such as pH, temperature, buffer composition, and peptide concentration, [13][14][15][16][17][18][19][20] as well as upon the hydrophobicity or hydrophilicity of the substrate. 21 Although the mechanism underlying the aggregation of Aβ peptides in vitro has been the subject of numerous investigations over the past two decades, many fundamental questions remain unanswered.…”