Design of peptides that form amyloid-like fibrils capturing amyloid beta1-42 peptides

Amyloid beta-peptide (Abeta) plays a critical role in Alzheimer's disease (AD). The monomeric state of Abeta can self-assemble into oligomers, protofibrils, and amyloid fibrils. Since the fibrils and soluble oligomers are believed to be responsible for AD, the construction of molecules capable of capturing these species could prove valuable as a means of detecting these potentially toxic species and of providing information pertinent for designing drugs effective against AD. To this aim, we have designed short peptides with various hydrophobicities based on the sequence of Abeta14-23, which is a critical region for amyloid fibril formation. The binding of the designed peptides to Abeta and the amplification of the formation of peptide amyloid-like fibrils coassembled with Abeta are elucidated. A fluorescence assay utilizing thioflavin T, known to bind specifically to amyloid fibrils, revealed that two designed peptides (LF and VF, with the leucine and valine residues, respectively, in the hydrophobic core region) could form amyloid-like fibrils effectively by using mature Abeta1-42 fibrils as nuclei. Peptide LF also coassembled with soluble Abeta oligomers into peptide fibrils. Various analyses, including immunostaining with gold nanoparticles, enzyme-linked immunosorbent assays, and size-exclusion chromatography, confirmed that the LF and VF peptides formed amyloid-like fibrils by capturing and incorporating Abeta1-42 aggregates into their peptide fibrils. In this system, small amounts of mature Abeta1-42 fibrils or soluble oligomers could be transformed into peptide fibrils and detected by amplifying the amyloid-like fibrils with the designed peptides.     

Design of Multicomponent Peptide Fibrils with Ordered and Programmable Compositional Patterns

Advanced applications of biomacromolecular assemblies require a stringent degree of control over molecular arrangement, which is a challenge to current synthetic methods. Here we used a neighbor-controlled patterning strategy to build multicomponent peptide fibrils with an unprecedented capacity to manipulate local composition and peptide positions. Eight peptides were designed to have regulable nearest neighbors upon co-assembly, which, by simulation, afforded 412 different patterns within fibrils, with varied compositions and/or peptide positions. The fibrils with six prescribed patterns were experimentally constructed with high accuracy. The controlled patterning also applies to functionalities appended to the peptides, as exemplified by arranging carbohydrate ligands at nanoscale precision for protein recognition. This study offers a route to molecular editing of inner structures of peptide assemblies, prefiguring the uniqueness and richness of patterning-based material design.     

Silica Nanowires Templated by Amyloid‐like Fibrils

Many peptides self‐assemble to form amyloid fibrils. We previously explored the sequence propensity to form amyloid using variants of a designed peptide with sequence KFFEAAAKKFFE. These variant peptides form highly stable amyloid fibrils with varied lateral assembly and are ideal to template further assembly of non‐proteinaceous material. Herein, we show that the fibrils formed by peptide variants can be coated with a layer of silica to produce silica nanowires using tetraethyl‐orthosilicate. The resulting nanowires were characterized using electron microscopy (TEM), X‐ray fiber diffraction, FTIR and cross‐section EM to reveal a nanostructure with peptidic core. Lysine residues play a role in templating the formation of silica on the fibril surface and, using this library of peptides, we have explored the contributions of lysine as well as arginine to silica templating, and find that sequence plays an important role in determining the physical nature and structure of the resulting nanowires.     

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.     

Creating an Amyloid ‘Kaleidoscope’ Using Short Iodinated Peptides

Amyloid fibrils formed by peptides with different sequences exhibit diversified morphologies, material properties and activities, making them valuable for developing functional bionanomaterials. However, the molecular understanding underlying the structural diversity of peptide fibrillar assembly at atomic level is still lacking. In this study, by using cryogenic electron microscopy, we first revealed the structural basis underlying the highly reversible assembly of ¹GFGGNDNFG⁹ (referred to as hnRAC1) peptide fibril. Furthermore, by installing iodine at different sites of hnRAC1, we generated a collection of peptide fibrils with distinct thermostability. By determining the atomic structures of the iodinated fibrils, we discovered that iodination at different sites of the peptide facilitates the formation of diverse halogen bonds and triggers the assembly of entirely different structures of iodinated fibrils. Finally, based on this structural knowledge, we designed an iodinated peptide that assembles into new atomic structures of fibrils, exhibiting superior thermostability, that aligned with our design. Our work provides an in-depth understanding of the atomic-level processes underlying the formation of diverse peptide fibril structures, and paves the way for creating an amyloid “kaleidoscope” by employing various modifications and peptide sequences to fine-tune the atomic structure and properties of fibrillar nanostructures.     

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.     

Effect of lysine-28 side-chain acetylation on the nanomechanical behavior of alzheimer amyloid beta25-35 fibrils

Amyloid fibrils are self-associating filamentous structures formed from the 39- to 42-residue-long amyloid beta peptide (Abeta peptide). The deposition of Abeta fibrils is one of the most important factors in the pathogenesis of Alzheimer's disease. Abeta25-35 is a fibril-forming peptide that is thought to represent the biologically active, toxic form of the full-length Abeta peptide. We have recently shown that beta sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition, and the unzipping forces differ in fibrils composed of different peptides. In the present work, we explored the effect of epsilon-amino acetylation of the Lys28 residue on the magnitude of the unzipping force of Abeta25-35 fibrils. Although the gross structure of the Lys28-acetylated (Abeta25-35_K28Ac) and wild-type Abeta25-35 (Abeta25-35wt) fibrils were similar, as revealed by atomic force microscopy, the fundamental unzipping forces were significantly lower for Abeta25-35_K28Ac (20 +/- 4 pN SD) than for Abeta25-35wt (42 +/- 9 pN SD). Simulations based on a simple two-state model suggest that the decreased unzipping forces, caused most likely by steric constraints, are likely due to a destabilized zippered state of the fibril.     

Enhanced Fibril Fragmentation of N‐Terminally Truncated and Pyroglutamyl‐Modified Aβ Peptides

N‐terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aβ peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aβ fibrils against mechanical stress and fragmentation and explains the high propensity of pE‐modified peptides to form small and particularly toxic aggregates.     

Encapsulation and NMR on an aggregating peptide before fibrillogenesis

The early stages of peptide and protein aggregation include the formation of soluble oligomers, some of which may be cytotoxic. There is a paucity of structural information on these oligomers, however, because they are temporally unstable and tend to aggregate further into insoluble protofibrils and fibrils. To obtain structural information on soluble oligomers, we have developed a procedure for encapsulating a fibril-forming peptide, Peptide 1 (NH2-SDDYYYGFGSNKFGRPRDD-COOH), in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine single bilayer vesicles (POPC SBVs). We also encapsulated a non-fibril forming peptide, Peptide 2 (NH2-EEWEE-COOH), in POPC SBVs. The nominal concentration of Peptide 1 in the resulting 40 nm diameter SBVs was 2.4 +/- 0.1 mM, well above the concentration at which Peptide 1 forms fibrils. We demonstrated that these peptides had indeed been encapsulated by measuring longitudinal relaxation times (T1) in the presence and absence of a paramagnetic substance, 1 mM Gd-EDTA, by NMR spectroscopy. When the peptides were free in solution, they showed the expected shortening of T1 times and broadening of NMR peaks. In contrast, peptide encapsulated in POPC SBVs were shielded from the effects of Gd-EDTA and showed preservation of T1 values and NMR line widths. To demonstrate that encapsulation inhibits fibril formation, we measured one-dimensional proton (1D-1H) NMR spectra of the peptides in solution, and of the encapsulated peptides immediately after encapsulation, and 4 days after encapsulation, because Peptide 1 forms fibrils within 1 day. A 2.8 mM solution of Peptide 1 shows the loss of NMR signal expected for a fibrillizing peptide. In contrast, the 1D-1H spectra of encapsulated Peptide 1 measured immediately after encapsulation and 4 days after encapsulation were essentially identical, with preservation of line width at 4 days, i.e., well within the time frame of most high-resolution NMR experiments. Encapsulation may provide a means to obtain high-resolution NMR data on unstable soluble oligomers of peptides implicated in amyloidoses such as Alzheimer's Disease and provide the first detailed structural information about these possibly cytotoxic species that have hitherto been inaccessible to analysis.     

Thermodynamics of peptide dimer formation

The Replica Exchange Statistical Temperature Molecular Dynamics algorithm is used to study the equilibrium properties of a peptide monomer and dimer and the thermodynamics of peptide dimer formation. The simulation data are analyzed by the Statistical Temperature Weighted Histogram Analysis Method. Each 10-residue peptide is represented by a coarse-grained model with hydrophobic side chains and has an α-helix as its minimum energy configuration. It is shown that the configurational behavior of the dimer can be divided into four regions as the temperature increases: two folded peptides; one folded and one unfolded peptide; two unfolded peptides; and two spatially separated peptides. Two important phenomena are discussed: in the dimer, one peptide unfolds at a lower temperature than the isolated monomer and the other peptide unfolds at a higher temperature than the isolated monomer. In addition, in the temperature region where one peptide is folded and the other unfolded, the unfolded peptide adopts an extended structure that minimizes the overall surface area of the aggregate. It is suggested that combination of destabilization due to aggregation and the resulting extended configuration of the destabilized peptide could have implications for nucleating β-sheet structures and the ultimate formation of fibrils.     

小热休克蛋白Mj HSP16.5对多肽纤维生长的抑制及对成熟纤维的解聚作用

包括老年痴呆症在内的许多疾病与蛋白质或多肽的淀粉样聚集(纤维化)有关. 由于这类疾病的机制尚不清楚, 因此还没有有效的预防和治疗手段. 研究各种因素如小热休克蛋白对蛋白质或多肽淀粉样聚集的影响对开发防治相关疾病的药物具有重要意义. 甲状腺素运载蛋白(TTR)及其突变体很容易形成淀粉样纤维, 并与多种疾病相关. Mj HSP16.5是一种来源于嗜热古细菌ethanococcus jannaschii的小热休克蛋白, 它在酸性条件下具有非常高的分子伴侣活性. 本文研究了Mj HSP16.5对WTTR肽(在N端添加了色氨酸的TTR 105-115片段, 序列为WYTIAALLSPYS)纤维化的影响, 发现Mj HSP16.5能够显著地抑制WTTR肽纤维的生长, 且在Mj HSP16.5存在下, WTTR肽形成的纤维比正常条件下形成的要显著细小. 尤其是Mj HSP16.5还可以使已经成熟的WTTR肽纤维解离. 结果表明, Mj HSP16.5抑制多肽纤维的机理可能在于其能够与多肽纤维及纤维种子结合.     

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.     

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.     

Time-averaged predictions of folded and misfolded peptides using a reduced physicochemical model

Energy-based methods for calculating time-averaged peptide structures are important for rational peptide design, for defining local structure propensities in large protein chains, and for exploring the sequence determinants of amyloid formation. High-end methods are currently too slow to be practicable, and will remain so for the foreseeable future. The challenge is to create a method that runs quickly on limited computer resources and emulates reality sufficiently well. We have developed a simplified off-lattice protein model, incorporating semi-empirical physicochemical potentials, and combined it with an efficient Monte Carlo method for calculating time-averaged peptide structures. Reasonably accurate predictions are found for a set of small alpha-helical and beta-hairpin peptides, and we demonstrate a potential application in measuring local structure propensities in protein chains. Time-averaged structures have also been calculated for a set of small peptides known to form beta-amyloid fibrils. The simulations were of three interacting peptides, and in each case the time-averaged structure describes a three-stranded beta-sheet. The performance of our method in measuring the propensities of small peptides to self-associate into possible prefibrillar species compares favorably with more detailed and CPU-intensive approaches.     

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.     

A Mobile Precursor Determines Amyloid-β Peptide Fibril Formation at Interfaces

The aggregation of peptides into amyloid fibrils plays a crucial role in various neurodegenerative diseases. While it has been generally recognized that fibril formation in vivo may be greatly assisted or accelerated by molecular surfaces, such as cell membranes, little is known about the mechanism of surface-mediated fibrillation. Here we study the role of adsorbed Alzheimer's amyloid-β peptide (Aβ42) on surface-mediated fibrillation using polymer coatings of varying hydrophobicity as well a supported lipid bilayer membrane. Using single molecule fluorescent tracking and atomic force microscopy imaging, we show that weakly adsorbed peptides with two-dimensional diffusivity are critical precursors to fibril growth on surfaces. This growth mechanism is inhibited on the highly hydrophilic surface where the surface coverage of adsorbed peptides is negligible or on the highly hydrophobic surface where the diffusion constant of the majority of adsorbed peptides is too low. Physical properties that favor weakly adsorbed peptides with sufficient translational mobility can locally concentrate peptide molecules on the surface and promote inter-peptide interaction via two-dimensional confinement, leading to fibrillation at Aβ peptide concentration many orders of magnitude below the critical concentration for fibrillation in the bulk solution.     

Amyloidosis of Alzheimer's Abeta peptides: solid-state nuclear magnetic resonance, electron paramagnetic resonance, transmission electron microscopy, scanning transmission electron microscopy and atomic force microscopy studies

Aggregation cascade for Alzheimer's amyloid-beta peptides, its relevance to neurotoxicity in the course of Alzheimer's disease and experimental methods useful for these studies are discussed. Details of the solid-phase peptide synthesis and sample preparation procedures for Alzheimer's beta-amyloid fibrils are given. Recent progress in obtaining structural constraints on Abeta-fibrils from solid-state NMR and scanning transmission electron microscopy (STEM) data is discussed. Polymorphism of amyloid fibrils and oligomers of the 'Arctic' mutant of Abeta(1-40) was studied by (1)H,(13)C solid-state NMR, transmission electron microscopy (TEM) and atomic force microscopy (AFM), and a real-time aggregation of different polymorphs of the peptide was observed with the aid of in situ AFM. Recent results on binding of Cu(II) ions and Al-citrate and Al-ATP complexes to amyloid fibrils, as studied by electron paramagnetic resonance (EPR) and solid-state (27)Al NMR techniques, are also presented.     

The organization of aromatic side groups in an amyloid fibril probed by solid-state 2H and 19F NMR spectroscopy

Some 25 diseases are associated with proteins and peptides that assemble into amyloid fibrils composed of beta-strands connected by hydrogen bonds oriented parallel to the fiber long axis. There is mounting evidence that amyloid formation involves specific interactions between amino acid side groups, which bring together beta-sheets to form layers with buried and exposed faces. This work demonstrates how a combination of solid-state 2H and 19F NMR experiments can provide constraints on fibril architecture by probing the environment and spatial organisation of aromatic side groups. It is shown that phenylalanine rings within fibrils formed by a decapeptide fragment of the islet amyloid polypeptide, amylin, are highly motionally restrained and are situated within 6.5 A of one another. Taken together with existing structural constraints for this peptide, these results are consistent with a fibril architecture that comprises layers of two or more beta-sheets, with the aromatic residues facing into the inter-sheet space and possibly engaged in pi-pi interactions. The methods presented will be of general utility in exploring the architecture of fibrils of larger, full-length peptides and proteins, including amylin itself.     

Templated growth of hybrid structures at the peptide-peptide interface

This study describes the use of peptide vesicular platforms for the templated growth of fibrillar structures to craft hybrids that retain the gross morphological features of two discreet self-assembled peptides. A synthetic triskelion peptide, which results in the rapid emergence of self-assembled spherical structures, was employed as a template. Addition of either one of two different peptides, both of which form long filamentous structures when co-incubated with the triskelion solution, affords hybrids that retain the gross morphology of both the spherical and filamentous structures. It is surmised that this process is aided by hydrogen bonding and the interdigitation of aromatic residues, which leads to the growth of hybrid structures. We believe that observations concerning the surface-assisted growth of peptide fibrils and tubular structures from vesicular platforms may have ramifications for the design and development of peptide-based hybrid materials with controlled hierarchical structures.     

Aggregation of Beta‐Amyloid Peptides Proximal to Zwitterionic Lipid Bilayers

One of the hallmarks of Alzheimers disease is the deposition of amyloid plaques, which consist of β‐amyloid (Aβ) peptides in fibrillar states. Nonfibrillar Aβ aggregates have been considered as an important intermediate in the pathway of fibrillization, but little is known about the formation mechanism. The on‐pathway β‐sheet intermediates of Aβ₄₀ peptides can be trapped by incubating the peptides in liposomes formed by zwitterionic lipids. The aggregates of Aβ₄₀ peptides have been prepared at a peptide concentration of less than 10 μm . Solid‐state NMR spectroscopy data show that the backbone conformation of the aggregates is almost identical to that of the fibrils formed in free solution. In contrast to anionic lipids, zwitterionic lipids, which are typical of neuronal soma, did not induce any significant conformational difference in Aβ₄₀ fibrils. This liposome–Aβ system may serve as a useful model to study the fibril formation mechanism.