THE ULTRASTRUCTURE OF FLAGELLAR FIBRILS

The tips of rat sperm tails were slightly frayed by mechanical agitation, thus exposing the fibrils, which were then studied by electron microscopy after negative staining. Only the fibrils survived this treatment. Each fibril proved to be a cylinder with a hollow core. The walls of the cylinders were made up of 10 longitudinally oriented filaments. The filaments had a markedly beaded appearance, with a repeating period of 88 A. The filament thickness (bead width) was approximately 35 to 40 A. Beads of neighboring filaments were in register with each other so that cross-linking bound the filaments together to complete the wall structure of each fibril. The center-to-center spacing from one filament to the next was 55 to 60 A. The periodicity and the diameters of the filaments make it unlikely that the filaments are related to either actin or myosin. From the way the fibrils kinked, it can be inferred that they possessed considerable mechanical strength. It is consistent with present knowledge that fibrils of the mitotic apparatus may have the same basic structure as the flagellar fibrils. Under some circumstances, pairs of fibrils separated from one another along their length, except at their extreme tips. It was apparent that there was special bridging material to be found there. In other preparations, however, the paired fibrils remained together, indicating a powerful coupling mechanism.     

Amyloid-like fibril formation of co-chaperonin GroES: nucleation and extension prefer different degrees of molecular compactness

The molecular chaperone GroES, together with GroEL from Escherichia coli, is the best characterized protein of the molecular chaperone family. Here, we report on the in vitro formation of GroES amyloid-like fibrils and the mechanism of formation. When incubated for several weeks at neutral pH in the presence of the denaturant guanidine hydrochloride, GroES formed a typical amyloid fibril; unbranched, twisted, and extended filaments stainable by thioflavin T and Congo red. GroES fibril formation was accelerated by the addition of preformed fibril seeds, in accordance with a nucleation-extension mechanism. Interestingly, whereas the spontaneous formation of GroES fibrils was favored in the structural transition region of GroES dissociation/unfolding, the extension of fibrils from preformed fibril seeds was favored in the region corresponding to an expanded molecular state. We concluded that the two stages of GroES fibril formation prefer different molecular states of the same protein. The significance of this preference is discussed.     

Concentration dependence of alpha-synuclein fibril length assessed by quantitative atomic force microscopy and statistical-mechanical theory

The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human α-synuclein on the mean length of mature α-synuclein fibrils, which are associated with Parkinson's disease. We determine that the critical initial concentration, below which low-molecular-weight species dominate and above which fibrils are the dominant species, lies at ∼15 μM, in good agreement with earlier measurements using biochemical methods. In the concentration regime where fibrils dominate, we find that their mean length increases with initial concentration. These results correspond well to the qualitative predictions of a recent statistical-mechanical model of amyloid fibril formation. In addition, good quantitative agreement of the statistical-mechanical model with the measured mean fibril length as a function of initial protein concentration, as well as with the fibril length distributions for several protein concentrations, is found for reasonable values of the relevant model parameters. The comparison between theory and experiment yields, for the first time to our knowledge, an estimate of the magnitude of the free energies associated with the intermolecular interactions that govern α-synuclein fibril formation.     

Intrinsic linear heterogeneity of amyloid β protein fibrils revealed by higher resolution mass-per-length determinations

Amyloid β 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 dark-field and transmission electron microscopy in a preparation of seeded amyloid β 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 mass-per-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.     

Tip-mediated fusion involving unipolar collagen fibrils accounts for rapid fibril elongation, the occurrence of fibrillar branched networks in skin and the paucity of collagen fibril ends in vertebrates.

Collagen fibrils are the principal source of mechanical strength of connective tissues such as tendon, skin, cornea, cartilage and bone. The ability of these tissues to withstand tensile forces is directly attributable to the length and diameter of the fibrils, and to interactions between individual fibrils. Although electron microscopy studies have provided information on fibril diameters, little is known about the length of fibrils in tissue and how fibrils interact with each other. The question of fibril length has been difficult to address because fibril ends are rarely observed in cross-sections of tissue. The paucity of fibril ends, or tips, has led to controversy about how long individual fibrils might be and how the fibrils grow in length and diameter. This review describes recent discoveries that are relevant to these questions. We now know that vertebrate collagen fibrils are synthesised as short (1-3 microm) early fibrils that fuse end-to-end in young tissues to generate very long fibrils. The diameter of the final fibril is determined by the diameter of the collagen early fibrils. During a late stage of tissue assembly fibril tips fuse to fibril shafts to generate branched networks. Of direct relevance to fibril fusion is the fact that collagen fibrils can be unipolar or bipolar, depending on the orientation of collagen molecules in the fibril. Fusion relies on: (1) specific molecular interactions at the carboxyl terminal ends of unipolar collagen fibrils; and (2) the insulator function of small proteoglycans to shield the surfaces of fibrils from inappropriate fusion reactions. The fusion of tips to shafts to produce branched networks of collagen fibrils is an elegant mechanism to increase the mechanical strength of tissues and provides an explanation for the paucity of fibril tips in older tissue.     

Nonpolar substitution at the C-terminus of the prion protein, a mimic of the glycosylphosphatidylinositol anchor, partially impairs amyloid fibril formation

In contrast to most amyloidogenic proteins or peptides that do not contain any significant posttranslational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant nonpeptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with an N-myristoylamidomaleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core but controls the mode of lateral assembly of filaments into higher order fibrils.     

Mechanisms of protein fibril formation: nucleated polymerization with competing off-pathway aggregation

The formation of protein fibrils, and in particular amyloid fibrils, underlies many human diseases. Understanding fibril formation mechanisms is important for understanding disease pathology, but fibril formation kinetics can be complicated, making the relationship between experimental observables and specific mechanisms unclear. Here we examine one often-proposed fibril formation mechanism, nucleated polymerization with off-pathway aggregation. We use the characteristics of this mechanism to derive three tests that can be performed on experimental data to identify it. We also find that this mechanism has an especially striking feature: although increasing protein concentrations generally cause simple nucleated polymerizations to reach completion faster, they cause nucleated polymerizations with off-pathway aggregation to reach completion more slowly when the protein concentration becomes too high.     

Two type XII-like collagens localize to the surface of banded collagen fibrils

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.     

A single mutation induces amyloid aggregation in the alpha-spectrin SH3 domain: analysis of the early stages of fibril formation

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.     

N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer

The conformational properties of soluble α-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated α-synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N-terminal residues, as well as smaller long-range changes in residues 28-31, 43-46, and 50-66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated α-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular weight species, but does alter the conformational distributions of monomeric α-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.     

Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation

The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1–42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril–fibril assembly can play in the deposition of aggregation-prone peptides.     

Polymerization of the bacterial elongation factor for protein synthesis, EF-Tu.

The bacterial elongation factor for protein synthesis, EF-Tu, polymerizes into fibrils at pH 6.0. These fibrils are 0.7 microM in diameter, at least 200 microns in length, and are positively birefringent. Electron microscopic observations of negatively stained images demonstrates that the EF-Tu fibrils consist of bundles of individual filaments, approximately 5nm in diameter, aligned parallel to the long axis of the fibril. Polymerized EF-Tu exchanges nucleotide rapidly and interacts with the other elongation factor, EF-Ts. The antibiotic kirromycin induces the polymerization of EF-Tu into fibrils and even larger structures under nonpolymerizing conditions.     

UV-light exposed prion protein fails to form amyloid fibrils

Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.     

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-2₁ screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.