Cytochrome display on amyloid fibrils

Protein amyloid fibrils can be functionalized by coating the core protofilament with high concentrations of proteins and enzymes. This can be done elegantly by appending a functional domain to an amyloidogenic protein monomer, then assembling the monomers into a fibril. To display an array of biologically functional porphyrins on the surface of protein fibrils, we have fused the sequence of the small, soluble cytochrome b562 to an SH3 dimer sequence that can form classical amyloid fibrils rapidly under well-defined conditions. The resulting fusion protein also forms amyloid fibrils and, in addition, binds metalloporphyrins, at half of the porphyrin binding sites as shown by UV-vis and NMR spectroscopies. Once metalloporphyrins are bound to the fibrils, the resulting holo-cytochrome domains are spectroscopically identical to the wild type cytochrome. The concentration of metalloporphyrins on a saturated fibril is estimated to be of the order of approximately 20 mM, suggesting that they could be interesting systems for applications in nanotechnology.     

A β-solenoid model of the Pmel17 repeat domain: insights to the formation of functional amyloid fibrils

Pmel17 is a multidomain protein involved in biosynthesis of melanin. This process is facilitated by the formation of Pmel17 amyloid fibrils that serve as a scaffold, important for pigment deposition in melanosomes. A specific luminal domain of human Pmel17, containing 10 tandem imperfect repeats, designated as repeat domain (RPT), forms amyloid fibrils in a pH-controlled mechanism in vitro and has been proposed to be essential for the formation of the fibrillar matrix. Currently, no three-dimensional structure has been resolved for the RPT domain of Pmel17. Here, we examine the structure of the RPT domain by performing sequence threading. The resulting model was subjected to energy minimization and validated through extensive molecular dynamics simulations. Structural analysis indicated that the RPT model exhibits several distinct properties of β-solenoid structures, which have been proposed to be polymerizing components of amyloid fibrils. The derived model is stabilized by an extensive network of hydrogen bonds generated by stacking of highly conserved polar residues of the RPT domain. Furthermore, the key role of invariant glutamate residues is proposed, supporting a pH-dependent mechanism for RPT domain assembly. Conclusively, our work attempts to provide structural insights into the RPT domain structure and to elucidate its contribution to Pmel17 amyloid fibril formation.     

Peptide Self-Assembly into Amyloid Fibrils at Hard and Soft Interfaces—From Corona Formation to Membrane Activity

Peptides and proteins are exposed to a variety of interfaces in a physiological environment, such as cell membranes, protein nanoparticles (NPs), or viruses. These interfaces have a significant impact on the interaction, self-assembly, and aggregation mechanisms of biomolecular systems. Peptide self-assembly, particularly amyloid fibril formation, is associated with a wide range of functions; however, there is a link with neurodegenerative diseases, such as Alzheimer's disease. This review highlights how interfaces affect peptide structure and the kinetics of aggregation leading to fibril formation. In nature, many surfaces are nanostructures, such as liposomes, viruses, or synthetic NPs. Once exposed to a biological medium, nanostructures are coated with a corona, which then determines their activity. Both accelerating and inhibiting effects on peptide self-assembly have been observed. When amyloid peptides adsorb to a surface, they typically concentrate locally, which promotes aggregation into insoluble fibrils. Starting from a combined experimental and theoretical approach, models that allow for a better understanding of peptide self-assembly near hard and soft matter interfaces are introduced and reviewed. Research results from recent years are presented and relationships between biological interfaces, such as membranes and viruses, and amyloid fibril formation are proposed.     

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105–115) Amyloid Protofibrils

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end‐capping effects have been conducted to understand amyloid fibril development. However, atomic‐level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end‐capping conditions.     

Adsorption-induced fibronectin aggregation and fibrillogenesis

Fibronectin (Fn), a high molecular weight glycoprotein, is a central element of extracellular matrix architecture that is involved in several fundamental cell processes. In the context of bone biology, little is known about the influence of the mineral surface on fibronectin supramolecular assembly. We investigate fibronectin morphological properties induced by its adsorption onto a model mineral matrix of hydroxyapatite (HA). Fibronectin adsorption onto HA spontaneously induces its aggregation and fibrillation. In some cases, fibronectin fibrils are even found connected into a dense network that is close to the matrix synthesized by cultured cells. Fibronectin adsorption-induced self-assembly is a time-dependant process that is sensitive to bulk concentration. The N-terminal domain of the protein, known to be implicated in its self-association, does not significantly inhibit the protein self-assembly while increasing ionic strength in the bulk alters both aggregation and fibrillation. The addition of a non-ionic surfactant during adsorption tends to promote aggregation with respect to fibrillation. Ultimately, fibronectin fibrils appear to be partially structured like amyloid fibrils as shown by thioflavine T staining. Taken together, our results suggest that there might be more than one single organization route involved in fibronectin self-assembly onto hydroxyapatite. The underlying mechanisms are discussed with respect to Fn conformation, Fn/surface and Fn/Fn interactions, and a model of fibronectin fibrillogenesis onto hydroxyapatite is proposed.     

Assembly of Alzheimer's Aβ peptide into nanostructured amyloid fibrils

The self-assembly of β-amyloid (Aβ) peptide into highly ordered amyloid fibril structures represents one of the pathological hallmarks of Alzheimer's disease. This process leads to the transient stabilization of ordered or disordered intermediates, which are thought to act as the main pathogenic culprits in neurodegenerative amyloid disease. This review describes recent results from different biophysical techniques, ranging from structure determination to single-particle methods by which the outgrowth of individual fibrils can be followed, and it explains their contributions towards understanding the mechanism of assembly. Finally, we will outline emerging methods and molecules to specifically interfere with the assembly and pathogenic impact of Aβ peptide.     

Morphology and persistence length of amyloid fibrils are correlated to peptide molecular structure

The formation of amyloid fibrils is a self-assembly process of peptides or proteins. The superior mechanical properties of these fibrils make them interesting for materials science but constitute a problem in amyloid-related diseases. Amyloid structures tend to be polymorphic, and their structure depends on growth conditions. To understand and control the assembly process, insights into the relation between the mechanical properties and molecular structure are essential. We prepared long, straight as well as short, worm-like β-lactoglobulin amyloid fibrils and determined their morphology and persistence length by atomic force microscopy (AFM) and the molecular conformation using vibrational sum-frequency generation (VSFG) spectroscopy. We show that long fibrils with near-100% β-sheet content have a 40-times higher persistence length than short, worm-like fibrils with β-sheet contents below 80%.     

Structural regulation of a peptide-conjugated graft copolymer: a simple model for amyloid formation

The self-assembly of peptides and proteins into beta-sheet-rich high-order structures has attracted much attention as a result of the characteristic nanostructure of these assemblies and because of their association with neurodegenerative diseases. Here we report the structural and conformational properties of a peptide-conjugated graft copolymer, poly(gamma-methyl-L-glutamate) grafted polyallylamine (1) in a water-2,2,2-trifluoroethanol solution as a simple model for amyloid formation. Atomic force microscopy revealed that the globular peptide 1 self-assembles into nonbranching fibrils that are about 4 nm in height under certain conditions. These fibrils are rich in beta-sheets and, similar to authentic amyloid fibrils, bind the amyloidophilic dye Congo red. The secondary and quaternary structures of the peptide 1 can be controlled by manipulating the pH, solution composition, and salt concentration; this indicates that the three-dimensional packing arrangement of peptide chains is the key factor for such fibril formation. Furthermore, the addition of carboxylic acid-terminated poly(ethylene glycol), which interacts with both of amino groups of 1 and hydrophobic PMLG chains, was found to obviously inhibit the alpha-to-beta structural transition for non-assembled peptide 1 and to partially cause a beta-to-alpha structural transition against the 1-assembly in the beta-sheet form. These findings demonstrate that the amyloid fibril formation is not restricted to specific protein sequences but rather is a generic property of peptides. The ability to control the assembled structure of the peptide should provide useful information not only for understanding the amyloid fibril formation, but also for developing novel peptide-based material with well-defined nanostructures.     

Protofilaments, filaments, ribbons, and fibrils from peptidomimetic self-assembly: implications for amyloid fibril formation and materials science

Deciphering the mechanism(s) of β-sheet mediated self-assembly is essential for understanding amyloid fibril formation and for the fabrication of polypeptide materials. Herein, we report a simple peptidomimetic that self-assembles into polymorphic β-sheet quaternary structures including protofilaments, filaments, fibrils, and ribbons that are reminiscent of the highly ordered structures displayed by the amyloidogenic peptides Aβ, calcitonin, and amylin. The distribution of quaternary structures can be controlled by and in some cases specified by manipulating the pH, buffer composition, and the ionic strength. The ability to control β-sheet-mediated assembly takes advantage of quaternary structure dependent pK(a) perturbations. Biophysical methods including analytical ultracentrifugation studies as well as far-UV circular dichroism and FT-IR spectroscopy demonstrate that linked secondary and quaternary structural changes mediate peptidomimetic self-assembly. Electron and atomic force microscopy reveal that peptidomimetic assembly involves numerous quaternary structural intermediates that appear to self-assemble in a convergent fashion affording quaternary structures of increasing complexity. The ability to control the assembly pathway(s) and the final quaternary structure(s) afforded should prove to be particularly useful in deciphering the quaternary structural requirements for amyloid fibril formation and for the construction of noncovalent macromolecular structures.     

Mass spectrometry and the amyloid problem—How far can we go in the gas phase?

A number of proteins are capable of converting from their soluble, monomeric form into highly-ordered, insoluble aggregates known as amyloid fibrils. In vivo, these fibrils, which accumulate in organs and tissues, are associated with a wide range of amyloid diseases for which there are currently no therapeutic solutions. The molecular details of the pathway from native monomer through oligomeric intermediates to the final amyloid fibril remain a challenging enigma. Over the past few years, mass spectrometry has been applied to investigate the various stages of amyloid fibril formation, and this report summarizes the key steps achieved to date.     

Polymer physics inspired approaches for the study of the mechanical properties of amyloid fibrils

Amyloid structures constitute a class of highly ordered nanomaterials formed by insoluble protein aggregates. These aggregates are characterized by a cross‐β structural motif in which β‐sheets are oriented perpendicular to the fibril axis and bound together by a dense hydrogen bonding network. Although they have been associated with several neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, amyloid fibrils have also been found in many physiologically beneficial roles, for instance in adhesives and hormone storage. Inspired by this natural occurrence of functional amyloid, the hierarchal self‐assembly of these structures has recently been used to develop artificial biomaterials for applications in medicine and nanotechnology. In order to realize the full potential of amyloids as functional materials, it is important to understand their fundamental mechanical properties. This review explores a range of experimental strategies to determine the mechanical properties of amyloid fibrils and discusses the results in the context of polymer physics concepts. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 281–292     

Natural protective amyloids

Amyloidoses are a group of diseases including neurodegenerative diseases like Alzheimer's disease and also type II diabetes, spongiform encephalopathies and many others, believed to be caused by protein aggregation and subsequent amyloid fibril formation. However, occasionally, living organisms exploit amyloid fibril formation, a property inherent into amino acid sequences, and perform specific physiological functions from amyloids, in differing biological contexts. Some of these functional amyloids are natural protective amyloids. Here, we review recent evidence on silkmoth chorion protein synthetic peptide-analogues that documents the function of silkmoth chorion, the major component of the eggshell, a structure with extraordinary physiological and mechanical properties, as a natural protective amyloid. Also, we briefly discuss the reported function of other natural, protective amyloids like fish chorion, the protein Pmel17 which forms amyloid fibrils that act as templates and accelerate the covalent polymerization of reactive small molecules into melanin, the hydrophobins and the antifreeze protein from winter flounder. Molecular self-assembly is becoming an increasingly popular route to new supramolecular structures and molecular materials and the inspiration for such structures is commonly derived from self-assembling systems in biology. Therefore, a careful examination of these studies may set the basis for the exploration of new routes for the formation of novel biocompatible polymeric structures with exceptional physico-chemical properties, for potentially new biomedical and industrial applications.     

Peptides derived from two dynamically disordered proteins self-assemble into amyloid-like fibrils

Short peptides derived from p14ARF and Hdm2 (14 and 15 amino acids in length, respectively), two cancer associated proteins, have been found to co-assemble into amyloid-like structures. Larger protein domains containing these peptide segments interact in cells and also undergo a disorder-to-order transition upon binding in vitro. In contrast to the association of beta-strand assemblies with amyloid diseases, the system described herein utilizes the formation of binary, extended beta-strands as a novel mechanism of biomolecular assembly. The beta-strand-containing fibrils formed from these peptides may allow the directed assembly of decorated fibrils with applications as biological nanostructures.     

A designed protein interface that blocks fibril formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures.     

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.     

De novo design of strand-swapped beta-hairpin hydrogels

De novo designed peptides, capable of undergoing a thermally triggered beta-strand-swapped self-assembly event leading to hydrogel formation were prepared. Strand-swapping peptide 1 (SSP1) incorporates an exchangeable beta-strand domain composed of eight residues appended to a nonexchangeable beta-hairpin domain. CD shows that, at pH 9 and temperatures less than 35 degrees C, this peptide adopts a random coil conformation, rendering it soluble in aqueous solution. On heating to 37 degrees C or greater, SSP1 adopts a beta-hairpin that displays an exchangeable beta-strand region. The exchangeable strand domain participates in swapping with the exchangeable domain of another peptide, affording a strand-swapped dimer. These dimers further assemble into fibrils that define the hydrogel. A second peptide (SSP2) containing an exchangeable strand composed of only four residues was also studied. Microscopy and scattering data show that the length of the exchangeable domain directly influences the fibril nanostructure and can be used as a design element to construct either twisted (SSP1) or nontwisted (SSP2) fibril morphologies. CD, FTIR, and WAXS confirm that both peptides adopt beta-sheet secondary structure when assembled into fibrils. Fibril dimensions, as measured by TEM, AFM, and SANS indicate a fibril diameter of 6.4 nm, a height of 6.0 nm, and a pitch of 50.4 nm for the twisted SSP1 fibrils. The nontwisted SSP2 fibrils are 6.2 nm in diameter and 2.5 nm in height. Oscillatory rheology, used to measure bulk hydrogel rigidity, showed that the gel composed of the nontwisted fibrils is more mechanically rigid (517 Pa at 6 rad/s) than the gel composed of twisted fibrils (367 Pa at 6 rad/s). This work demonstrates that beta-strand-swapping can be used to fabricate biomaterials with tunable fibril nanostructure and bulk hydrogel rheological properties.     

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In Vivo

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post‐translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.     

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In Vivo

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.     

Amyloid fibril formation and other aggregate species formed by human serum albumin association

Under in vitro solution conditions where the native state is destabilized, many proteins present an abnormal structure and metabolism associated with a strong tendency to self-aggregation into a polymeric amyloid fibril structure, suggesting that this ability is a generic feature of the polypeptide chains. Such structures play a key role in different pathogenesis of neurodegenerative diseases such as Alzheimer, Parkinson, or Creutzfeldt-Jakob. Here, we report the formation of amyloid fibrils in the plasma protein human serum albumin under different in vitro conditions monitored using a combination of spectrophotometric and microscopic techniques. Amyloid fibril formation, therefore, is also allowed in a protein with a high degree of structural complexity. We also infer from experimental data the existence of other protein aggregated species than fibrils, some of which seem to be formed by a structural rearrangement of the proper fibrils.