Ehud Gazit

A possible role for π-stacking in the self-assembly of amyloid fibrils

Amyloid fibril formation is assumed to be the molecular basis for a variety of diseases of unrelated origin. Despite its fundamental clinical importance, the mechanism of amyloid formation is not fully understood. When we analyzed a variety of short functional fragments from unrelated amyloid‐forming proteins, a remarkable occurrence of aromatic residues was observed. The finding of aromatic residues in diverse fragments raises the possibility that IT‐IT interactions may play a significant role in the molecular recognition and self‐assembly processes that lead to amyloid formation. This is in line with the well‐known central role of π‐stacking interactions in self‐assembly processes in the fields of chemistry and biochemistry. We speculate that the stacking interactions may provide energetic contribution as well as order and directionality in the self‐assembly of amyloid structures. Experimental data regarding amyloid formation and inhibition by short peptide analogs also support our hypothesis. The π‐stacking hypothesis suggests a new approach to understanding the self‐assembly mechanism that governs amyloid formation and indicates possible ways to control this process.—Gazit, E. A possible role for π‐stacking in the self‐assembly of amyloid fibrils. FASEB J. 16, 77–83 (2002)

Inhibition of Amyloid Fibril Formation by Polyphenols: Structural Similarity and Aromatic Interactions as a Common Inhibition Mechanism

The formation of well‐ordered fibrillar protein deposits is common to a large group of amyloid‐associated disorders. This group consists of several major human diseases such as Alzheimers disease, Parkinsons disease, prion diseases, and type II diabetes. Currently, there is no approved therapeutic agent directed towards the formation of fibrillar assemblies, which have been recently shown to have a key role in the cytotoxic nature of amyloidogenic proteins. One important approach in the development of therapeutic agents is the use of small molecules that specifically and efficiently inhibit the aggregation process. Several small polyphenol molecules have been demonstrated to remarkably inhibit the formation of fibrillar assemblies in vitro and their associated cytotoxicity. Yet, the inhibition mechanism was mostly attributed to the antioxidative properties of these polyphenol compounds. Based on several observations demonstrating that polyphenols are capable of inhibiting amyloid fibril formation in vitro, regardless of oxidative conditions, and in view of their structural similarities we suggest an additional mechanism of action. This mechanism is assuming structural constraints and specific aromatic interactions, which direct polyphenol inhibitors to the amyloidogenic core. This proposed mechanism is highly relevant for future de novo inhibitors‘ design as therapeutic agents for the treatment of amyloid‐associated diseases.

Self-assembled peptide nanostructures: the design of molecular building blocks and their technological utilization

In this tutorial review the process and applications of peptide self-assembly into nanotubes, nanospheres, nanofibrils, nanotapes, and other ordered structures at the nano-scale are discussed. The formation of well-ordered nanostructures by a process of self-association represents the essence of modern nanotechnology. Such self-assembled structures can be formed by a variety of building blocks, both organic and inorganic. Of the organic building blocks, peptides are among the most useful ones. Peptides possess the biocompatibility and chemical diversity that are found in proteins, yet they are much more stable and robust and can be readily synthesized on a large scale. Short peptides can spontaneously associate to form nanotubes, nanospheres, nanofibrils, nanotapes, and other ordered structures at the nano-scale. Peptides can also form macroscopic assemblies such as hydrogels with nano-scale order. The application of peptide building blocks in biosensors, tissue engineering, and the development of antibacterial agents has already been demonstrated.

Amyloids: Not Only Pathological Agents but Also Ordered Nanomaterials

Amyloid fibers constitute one of the most abundant and important naturally occurring self-associated assemblies. A variety of protein and peptide molecules with various amino acid sequences form these highly stable and well-organized assemblies under diverse conditions. These assemblies display phase states ranging from liquid crystals to rigid nanotubes. The potential applications of these supramolecular assemblies exceed those of synthetic polymers since the building blocks may introduce biological function in addition to mechanical properties. Here we review the structural characteristics of amyloidal supramolecular assemblies, their potential use as either natural or de novo designed sequences, and the range of applications that have been demonstrated so far.

Controlled patterning of aligned self-assembled peptide nanotubes.

Controlling the spatial organization of objects at the nanoscale is a key challenge in enabling their technological application1,2,3. Biomolecular assemblies are attractive nanostructures owing to their biocompatibility, straightforward chemical modifiability, inherent molecular recognition properties and their availability for bottom-up fabrication4,5,6,7,8,9,10,11,12,13,14,15,16. Aromatic peptide nanotubes are self-assembled nanostructures with unique physical and chemical stability and remarkable mechanical rigidity14,15,16. Their application in the fabrication of metallic nanowires and in the improvement of the sensitivity of electrochemical biosensors have already been demonstrated14,15,16,17. Here we show the formation of a vertically aligned nanoforest by axial unidirectional growth of a dense array of these peptide tubes. We also achieved horizontal alignment of the tubes through noncovalent coating of the tubes with a ferrofluid and the application of an external magnetic field. Taken together, our results demonstrate the ability to form a two-dimensional dense array of nanotube assemblies with either vertical or horizontal patterns.

Amyloid Fibril Formation by Pentapeptide and Tetrapeptide Fragments of Human Calcitonin

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH2-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH2-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH2-FNKF-COOH tetrapeptide, the NH2-DFN-COOH tripeptide, or the NH2-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.

Self-assembled arrays of peptide nanotubes by vapour deposition

The use of bionanostructures in real-world applications will require precise control over biomolecular self-assembly and the ability to scale up production of these materials. A significant challenge is to control the formation of large, homogeneous arrays of bionanostructures on macroscopic surfaces. Previously, bionanostructure formation has been based on the spontaneous growth of heterogenic populations in bulk solution. Here, we demonstrate the self-assembly of large arrays of aromatic peptide nanotubes using vapour deposition methods. This approach allows the length and density of the nanotubes to be fine-tuned by carefully controlling the supply of the building blocks from the gas phase. Furthermore, we show that the nanotube arrays can be used to develop high-surface-area electrodes for energy storage applications, highly hydrophobic self-cleaning surfaces and microfluidic chips.

Self-Assembled Fmoc-Peptides as a Platform for the Formation of Nanostructures and Hydrogels

Hydrogels are of great interest as a class of materials for tissue engineering, axonal regeneration, and controlled drug delivery, as they offer 3D interwoven scaffolds to support the growth of cells. Herein, we extend the family of the aromatic Fmoc-dipeptides with a library of new Fmoc-peptides, which include natural and synthetic amino acids with an aromatic nature. We describe the self-assembly of these Fmoc-peptides into various structures and characterize their distinctive molecular and physical properties. Moreover, we describe the fabrication of the bioactive RGD sequence into a hydrogel. This unique material offers new opportunities for developing cell-adhesive biomedical hydrogel scaffolds, as well as for establishing strategies to modify surfaces with bioactive materials.

Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool.

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self‐assembled amyloid fibrils are also involved in physiological processes in several micro‐organisms. Yet, the molecular basis for the recognition and self‐assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self‐assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid‐associated disorders.

Allostery and Intrinsic Disorder Mediate Transcription Regulation by Conditional Cooperativity

Regulation of the phd/doc toxin-antitoxin operon involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc, a phenomenon known as conditional cooperativity. The mechanism underlying this observed behavior is not understood. Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain, illustrating allosteric coupling between highly disordered and highly unstable domains. This allosteric effect also couples Doc neutralization to the conditional regulation of transcription. In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action. Our experiments provide the basis for understanding the mechanism of conditional cooperative regulation of transcription typical of toxin-antitoxin modules. This model may be applicable for the regulation of other biological systems.