Spider silk and amyloid fibrils: a structural comparison

Although spider silks have been studied for decades, the assembly properties of the underlying silk proteins have still not been unravelled. Previously, the detection of amyloid-like nanofibrils in the spider's silk gland suggested their involvement in the assembly process.Recombinantly produced spider silk also self-assembles into nanofibrils. In order to investigate the structural properties of such silk nanofibrils in more detail, they have been compared to amyloid-like fibrils to highlight structural similarities.     

Amyloid-like fibril-forming supramolecular β-sheets from a β-turn forming tripeptide containing non-coded amino acids: the crystallographic signature

The crystal structure of a terminally protected tripeptide Boc-Leu-Aib-β-Ala-OMe 1 containing non-coded amino acids reveals that it adopts a β-turn structure, which self-assembles to form a supramolecular β-sheet via non-covalent interactions. The SEM image of peptide 1 exhibits amyloid-like fibrillar morphology in the solid state.     

An amyloid-like fibril forming antiparallel supramolecular β-sheet from a synthetic tripeptide: a crystallographic signature

Single crystal X-ray diffraction studies of a terminally blocked tripeptide Boc-Leu(1)-Aib(2)-Leu(3)-OMe 1 demonstrates that it adopts a bend structure without any intramolecular hydrogen bond. Peptide 1 self-assembles to form a supramolecular antiparallel β-sheet structure by various non-covalent interactions including intermolecular hydrogen bonds in the crystal and it exhibits amyloid-like fibrillar morphology in the solid state.     

The role of the disulfide bond in amyloid-like fibrillogenesis in a model peptide system

Three terminally protected short peptides Bis[Boc-D-Leu1-Cys2-OMe] 1, Bis[Boc-Leu1-Cys2-OMe] and Bis[Boc-Val1-Cys2-OMe] 3 exhibit amyloid-like fibrillar morphology. Single crystal X-ray diffraction analysis of peptide 1 clearly demonstrates that it adopts an overall extended backbone molecular conformation that self-assembles to form an intermolecular hydrogen-bonded antiparallel supramolecular beta-sheet structure in crystals. Scanning electron microscopic (SEM) images, transmission electron microscopic (TEM) images and Congo red binding studies vividly demonstrate the amyloid-like fibril formation of peptides 1, 2 and 3. However, after reduction of the disulfide bridge of peptides 1, 2 and 3, three newly generated peptides Boc-D-Leu1-Cys2-OMe 4, Boc-Leu1-Cys2-OMe 5 and Boc-Val1-Cys2-OMe 6 are formed and all of them failed to form any kind of fibril under the same conditions, indicating the important role of the disulfide bond in amyloid-like fibrillogenesis in a peptide model system.     

beta-Sheet mediated self-assembly of dipeptides of omega-amino acids and remarkable fibrillation in the solid state

Single crystal X-ray diffraction studies show that the extended structure of dipeptide Boc-beta-Ala-m-ABA-OMe (m-ABA: meta-aminobenzoic acid) self-assembles in the solid state by intermolecular hydrogen bonding to create an infinite parallel beta-sheet structure. In dipeptide Boc-gamma-Abu-m-ABA-OMe (gamma-Abu: gamma-aminobutyric acid), two such parallel beta-sheets are further cross-linked by intermolecular hydrogen bonding through m-aminobenzoic acid moieties. SEM (scanning electron microscopy) studies reveal that both the peptides and form amyloid-like fibrils in the solid state. The fibrils are also found to be stained readily by Congo red, a characteristic feature of the amyloid fiber whose accumulation causes several fatal diseases such as Alzheimer's, prion-protein etc.     

An amyloid-like fibril-forming supramolecular cross-β-structure of a model peptide: a crystallographic insight

The peptide Boc-Val-Phe-OMe 1 bearing sequence similarity with the central hydrophobic cluster (CHC) of Alzheimer's Aβ(18-19) peptide self-assembles to produce amyloid-like straight unbranched fibrils as examined by atomic force microscopy and Congo red assay. Single crystal X-ray diffraction offers the atomic level structure of the supramolecular parallel β-sheet aggregation and antiparallel separation between layers (cross-β-structure).     

Intramolecular ditryptophan crosslinks enforce two types of antiparallel beta structures.

BACKGROUND: Two types of biaryl crosslinks can be formed with natural protein sidechains: ditryptophan and dityrosine. Biaryl crosslinks have the same topology as disulfide crosslinks, yet little is known about their effect on local peptide structure. RESULTS: Three ditryptophan-linked peptide dimers based on the sequence Ac-Leu-Trp-Ala-COX were prepared. The tripeptide dimer with -CONH(2) termini was too insoluble to study, but the tripeptide dimer with -COOMe termini crystallized from methanol/chloroform as an antiparallel beta-sheet. The tripeptide dimer with a -CONMe(2) termini adopted a slipped antiparallel beta structure in methanol/chloroform. CONCLUSIONS: These results suggest that intermolecular ditryptophan crosslinks that join the middle of peptide chains can confer a preference for antiparallel beta-sheet structure. The effect is most dramatic when both the inside and outside edges of the dimer can form hydrogen bonds as in the crystal structure of dimer 3b.     

Investigation of secondary structure elements by IR/UV double resonance spectroscopy: analysis of an isolated beta-sheet model system

An isolated beta-sheet model system is investigated in a molecular beam experiment by means of mass- and isomer-selective IR/R2PI double resonance spectroscopy as well as ab initio and DFT calculations. As the exclusive intermolecular assembly, a beta-sheet motif is formed by spontaneous dimerization of two isolated peptide molecules. This secondary structure is produced from the tripeptide model Ac-Val-Tyr(Me)-NHMe without any further environment to form the binding motif which is analyzed by both the characteristic amide A and I vibrations. The experimental and theoretical investigations yield the assignment to an antiparallel beta-sheet model. The result of this detailed spectroscopic analysis on an isolated beta-sheet model indicates that there are intrinsic properties of a beta-sheet structure which can be formed without a solvent or a peptidic environment.     

Genetic engineering combined with deep UV resonance Raman spectroscopy for structural characterization of amyloid-like fibrils

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.     

Stepwise Self-assembly of a Tripeptide from Molecular Dimers to Supramolecular β-sheets in Crystals and Amyloid-like Fibrils in the Solid State

The terminally protected tripeptide Boc–Ala(1)–Leu(2)–Ala(3)–OMe 1 forms antiparallel hydrogen-bonded dimers of two different conformers in the asymmetric unit and the individual dimers then self-associate to form supramolecular β-sheet structures in crystals and amyloid-like fibrils in the solid state.     

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.     

Self-assembled template-directed synthesis of one-dimensional silica and titania nanostructures

Mineralized biological materials such as shells, skeleton, and teeth experience biomineralization. Biomimetic materials exploit the biomineralization process to form functional organic-inorganic hybrid nanostructures. In this work, we mimicked the biomineralization process by the de novo design of an amyloid-like peptide that self-assembles into nanofibers. Chemically active groups enhancing the affinity for metal ions were used to accumulate silicon and titanium precursors on the organic template. The self-assembly process and template effect were characterized by CD, FT-IR, UV-vis, fluorescence, rheology, TGA, SEM, and TEM. The self-assembled organic nanostructures were exploited as a template to form high-aspect-ratio 1-D silica and titania nanostructures by the addition of appropriate precursors. Herein, a new bottom-up approach was demonstrated to form silica and titania nanostructures that can yield wide opportunities to produce high-aspect-ratio inorganic nanostructures with high surface areas. The materials developed in this work have vast potential in the fields of catalysis and electronic materials.     

Multiscale surface self-assembly of an amyloid-like peptide

We present the 2D self-assembly properties of an amyloid-like peptide (LSFDNSGAITIG-NH2) (i.e., LSFD) over a whole range of spatial scales. This peptide is known to adopt an amyloid-like behavior in water where it aggregates into fibrils. Monolayers of this 12 amino acid peptide were built by direct spreading and compression of an organic unstructured LSFD solution at the air/water interface. Investigation by infrared spectroscopy of the peptide secondary structure reveals beta-sheet formation at the water surface. As evidenced by Brewster angle microscopy, compression of the peptidic film results in the formation of large condensed domains. We used atomic force microscopy to show that these domains are made of rather monodisperse, elongated domains of monomolecular thickness, which are about 1 microm long and hundred of nanometers wide. These nanodomains can be compacted up to the formation of a homogeneous monolayer on the micrometer scale. These bidimensional structures appear as a surface-induced counterpart of the bulk amyloid fibrils that do not form at the air/water interface. These self-assembled peptide nanostructures are also very promising for building organized nanomaterials.     

The diastereomeric assembly of polylysine is the low-volume pathway for preferential formation of beta-sheet aggregates

The interaction of left- and right-handed polylysine chains (poly(D-lysine) and poly(L-lysine)) results in a dramatic increase in the propensity to form aggregated beta-sheet structure (and amyloid-like fibrils), which is reflected by an approximately 15 degrees C decrease of temperature of the alpha-helix-to-beta-sheet transition. While a relative volume expansion of 13-19 mL x mol(-1) accompanies the alpha-to-beta-transition in a single enantiomer, this does not hold true for the mixture, which, along with substantially more negative heat capacity changes, points to a lower solvent-entropy cost of the transition as the possible thermodynamic driving force of the diastereomeric aggregation. The underlying solvational mechanism may be one of the decisive factors responsible for the spontaneous protein aggregation in vivo and, as such, may shed new light on the molecular basis of amyloid-associated diseases.     

Structural analysis of alanine tripeptide with antiparallel and parallel beta-sheet structures in relation to the analysis of mixed beta-sheet structures in Samia cynthia ricini silk protein fiber using solid-state NMR spectroscopy

The structural analysis of natural protein fibers with mixed parallel and antiparallel beta-sheet structures by solid-state NMR is reported. To obtain NMR parameters that can characterize these beta-sheet structures, (13)C solid-state NMR experiments were performed on two alanine tripeptide samples: one with 100% parallel beta-sheet structure and the other with 100% antiparallel beta-sheet structure. All (13)C resonances of the tripeptides could be assigned by a comparison of the methyl (13)C resonances of Ala(3) with different [3-(13)C]Ala labeling schemes and also by a series of RFDR (radio frequency driven recoupling) spectra observed by changing mixing times. Two (13)C resonances observed for each Ala residue could be assigned to two nonequivalent molecules per unit cell. Differences in the (13)C chemical shifts and (13)C spin-lattice relaxation times (T(1)) were observed between the two beta-sheet structures. Especially, about 3 times longer T(1) values were obtained for parallel beta-sheet structure as compared to those of antiparallel beta-sheet structure, which could be explicable by the difference in the hydrogen-bond networks of both structures. This very large difference in T(1) becomes a good measure to differentiate between parallel or antiparallel beta-sheet structures. These differences in the NMR parameters found for the tripeptides may be applied to assign the parallel and antiparallel beta-sheet (13)C resonances in the asymmetric and broad methyl spectra of [3-(13)C]Ala silk protein fiber of a wild silkworm, Samia cynthia ricini.     

Self-assembling small molecules form nanofibrils that bind procaspase-3 to promote activation

Modulating enzyme function with small-molecule activators, as opposed to inhibitors, offers new opportunities for drug discovery and allosteric regulation. We previously identified a compound, called 1541, from a high-throughput screen (HTS) that stimulates activation of a proenzyme, procaspase-3, to generate mature caspase-3. Here we further investigate the mechanism of activation and report the surprising finding that 1541 self-assembles into nanofibrils exceeding 1 μm in length. These particles are an unanticipated outcome from an HTS that have properties distinct from standard globular protein aggregators. Moreover, 1541 nanofibrils function as a unique biocatalytic material that activates procaspase-3 via induced proximity. These studies demonstrate a novel approach for proenzyme activation through binding to fibrils, which may mimic how procaspases are naturally processed on protein scaffolds.     

Self-assembly and gelation of oxidized glutathione in organic solvents.

The oxidized disulfide form of the ubiquitous tripeptide glutathione (gamma-glu-cys-gly) (GSSG) is shown to produce transparent, thermoreversible gels in aqueous solutions of dimethyl sulfoxide, dimethylformamide, and methanol, at GSSG concentrations as low as 1.5 mM. The gels bind Congo Red and exhibit dramatic green birefringence when observed between crossed polarizers, characteristic of amyloid structures. By transmission electron microscopy, the gels appear to consist of a network of fibrous structures about 75 nm in diameter. Several structurally related peptides, including the glutathione isomer glu-cys-gly and the aspartyl analogue of glutathione (beta-asp-cys-gly), failed to produce gels under similar conditions. These results suggest that the interactions which produce gelation are highly specific and that the unusual peptide geometry introduced by gamma-glu-cys linkage is critical to the gelation behavior. (1)H NMR indicates solvent-dependent perturbation of the gamma-glutamyl alpha- and beta-protons and circular dichroism reveals a shift in the geometry of the disulfide bond under conditions producing gelation. We propose that in appropriate organic solvents, GSSG self-assembles into an extended network of beta-sheetlike structures capable of immobilizing bulk solvent. While obviously speculative, it is interesting to consider possible physiological consequences of glutathione self-recognition in such processes as abnormal protein aggregation and the thiol-disulfide exchange which is believed to participate in protein folding.     

Assembly of a trifunctional artificial peptide into an anti-parallel duplex with three Cu(II) cross-links

Analogous to self-assembly in natural DNA or proteins, we describe the synthesis of a heterofunctional artificial tripeptide that self-assembles into an antiparallel duplex by coordination of three Cu(II) ions. The tripeptide contains three pendant ligands, pyridine (py), methyl bipyridine (bpy), and terpyridine (tpy), in series on an aminoethylglycine (aeg) backbone. These ligands chelate three Cu(II) ions, forming two [Cu(tpy)(py)](2+) and one [Cu(bpy)(2)](2+) complexes, that cross-link two tripeptide strands to give a trimetallic supramolecular structure. The tripeptide and metal-linked tripeptide duplex are characterized with NMR spectroscopy, mass spectrometry, and analytical high performance liquid chromatography (HPLC). Spectrophotometric titrations are used to quantitatively examine the stoichiometry of binding. Together with electron paramagnetic resonance (EPR) spectroscopy, the identities of the Cu(II) complexes and their environments are examined. The EPR spectrum reveals a significant amount of coupling between metal centers compared to a dimetallic dipeptide analogue. EPR and UV-vis absorbance spectroscopy, together with molecular modeling, provide evidence that the tripeptide acts as a scaffold to hold the metal centers in close proximity.     

Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.     

Structural control of self-assembled nanofibers by artificial beta-sheet peptides composed of D- or L-isomer

A novel method for the control of peptide self-assembly has been developed by using synthetic triblock-type beta-sheet peptides composed of l- or d-amino acid, 1L and 1D, as building blocks. The peptides 1L and 1D self-assemble into beta-sheet nanofibers with left- and right-handed twists, respectively, under appropriate condition. On the other hand, the 1L/1D binary mixture was found to form only globular aggregates at the same condition. Thus, amyloid-like nanofiber formation and its nanostructure could be successfully regulated by the stereospecificity of the constituent peptide species.