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Biomedical subjects

Ehud Gazit

Publications and source records attributed to Ehud Gazit.

At least 19 recordsLinked to original sources
PubMed2006

Use of biomolecular templates for the fabrication of metal nanowires.

The nano-scale spatial organization of metallic and other inorganic materials into 1D objects is a key task in nanotechnology. Nano-scale fibers and tubes are very useful templates for such organization because of their inherent 1D organization. Fibrillar biological molecules and biomolecular assemblies are excellent physical supports on which to organize the inorganic material. Furthermore, these biological assemblies can facilitate high-order organization and specific orientation of inorganic structures by their utilization of highly specific biological recognition properties. In this minireview, I will describe the use of biomolecules and biomolecular assemblies, including DNA, proteins, peptides, and even viral particles, which are excellent templates for 1D organization of inorganic materials into wires. This ranges from simple attempts at electroless deposition on inert biological templates to the advanced use of structural motifs and specific protein-DNA interactions for nano-bio-lithography as well as the fabrication of multilayer organic and inorganic composites. The potential technological applications of these hybrid biological-inorganic assemblies will be discussed.

Amyloid↗
PubMed2006

The yefM-yoeB toxin-antitoxin systems of Escherichia coli and Streptococcus pneumoniae: functional and structural correlation.

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM(Spn)) and toxin (YoeB(Spn)) products. We showed that overproduction of YoeB(Spn) is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB(Spn)-mediated toxicity could be reversed by the cognate antitoxin, YefM(Spn), but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

Bacterial Toxins↗
PubMed2006

The molecular dynamics of assembly of the ubiquitous aortic medial amyloidal medin fragment.

In recent years there is an increased understanding of the molecular conformation of amyloid fibrils. However, much less is known about the early events that lead to the formation of these medically important assemblies. The clarification of these very important mechanistic details on the process may indicate directions towards the inhibition of the early stages of the assembly, where harmful species are most likely to form. Here, we study the dynamics of assembly of short amyloidogenic peptide fragments from the medin polypeptide. This polypeptide is of unique interest since amyloid deposits composed of medin are found almost in all the population above the age of 50. Twelve independent 50 ns long molecular dynamics simulations in explicit water have been run on peptide NH2-NFGSVQFV-COOH, the minimal recognition hexapeptide element, NH2-NFGSVQ-COOH, and several single-point mutants. In all cases a three-stranded polymeric beta-sheet was used as the basic unit from which fibrils can be formed. Our results clearly indicate the need of well-defined sequence and stereochemical constraints to allow the formation of stable well-ordered aggregates. One of the key findings is the need for the presence of a phenylalanine residue, but not other hydrophobic amino acids, in specific positions within the peptide. Taken together, the results are consistent with recent high-resolution structures of amyloid assemblies and provide unique insights into the dynamics of these structures.

Amino Acid Sequence↗
PubMed2006

From green bacteria to human dementia: a novel model for discovering amyloid assembly inhibitors.

The formation of amyloid assemblies is associated with major human disorders. Yet no therapeutic agents presently exist to control this process. In a recent paper, a new bacterial system is described that uses a fusion of the Alzheimer's disease beta-amyloid polypeptide to the GFP. The assay detects the formation of small, soluble amyloid intermediates associated with degenerative diseases. This assay allows the researchers to use high-throughput screening methods to find inhibitors of the formation of amyloid assemblies.

Amyloid↗
PubMed2006

Direct observation of the release of phenylalanine from diphenylalanine nanotubes.

The core recognition motif of the amyloidogenic beta-amyloid polypeptide is a dipeptide of phenylalanine. This dipeptide readily self-assembles to form discrete, hollow nanotubes with high persistence lengths. The simplicity of the nanotube formation, combined with ideal physical properties, make these nanotubes highly desirable for a range of applications in bionanotechnology. To fully realize the potential of such structures, it is first necessary to gain a comprehensive understanding of their chemical and physical properties. Previously, the thermal stability of these nanotubes has been investigated by electron microscopy. Here, we further our understanding of the structural stability of the nanotubes upon dry-heating using the atomic force microscope (AFM), and for the first time identify their degradation product utilizing time-of-flight secondary-ion mass spectrometry. We show that the nanotubes are stable at temperatures up to 100 degrees C, but on heating to higher temperatures begin to lose their structural integrity with an apparent collapse in tubular structure. With further increases in temperature up to and above 150 degrees C, there is a degradation of the structure of the nanotubes through the release of phenylalanine building blocks. The breakdown of structure is observed in samples that are either imaged at elevated temperatures or imaged following cooling, suggesting that once phenylalanine is lost from the nanotubes they are susceptible to mechanical deformation by the imaging AFM probe. This temperature-induced plasticity may provide novel properties for these peptide nanotubes, including possible applications as scaffolds and drug delivery devices.

Dipeptides↗
PubMed2006

Inhibition of amyloid fibril formation and cytotoxicity by hydroxyindole derivatives.

Gaining insight into the mechanism of amyloid fibril formation, the hallmark of multiple degenerative syndromes of unrelated origin, and exploring novel directions of inhibition are crucial for preventing disease development. Specific interactions between aromatic moieties were suggested to have a key role in the recognition and self-assembly processes leading to the formation of amyloid fibrils by several amyloidogenic polypeptides, including the beta-amyloid polypeptide associated with Alzheimer's disease. Our finding of the high-affinity molecular recognition and intense amyloidogenic potential of tryptophan-containing peptide fragments led to the hypothesis that screening for indole derivatives might lead to the identification of potential inhibitors of amyloid formation. Such inhibitors could mediate specific recognition processes without allowing further growth of the well-ordered amyloid chain. Using fluorescence spectroscopy, atomic force microscopy, and electron microscopy to screen 29 indole derivatives, we identified three potent inhibitors: indole-3-carbinol (I3C), 3-hydroxyindole (3HI), and 4-hydroxyindole (4HI). The latter, a simple low-molecular weight aromatic compound, was the most effective, completely abrogating not only the formation of aggregated structures by Abeta but also the cytotoxic activity of aggregated Abeta toward cultured cells. The results of this study provide further experimental support for the paradigm of amyloid inhibition by heteroaromatic interaction and point to indole derivatives as a simple molecular platform for the development of novel fibrillization inhibitors.

Amyloid↗
PubMed2006

Designed aromatic homo-dipeptides: formation of ordered nanostructures and potential nanotechnological applications.

Molecular self-assembly offers new routes for the fabrication of novel materials at the nano-scale. Peptide-based nanostructures represent nano-objects of particular interest, as they are biocompatible, can be easily synthesized in large amounts, can be decorated with functional elements and can be used in various biological and non-biological applications. We had previously revealed the formation of highly ordered tubular structures by the diphenylalanine peptide, the core recognition motif of Alzheimer's beta-amyloid polypeptide, due to specific aromatic interactions. We further confirmed this model and demonstrated that a non-charged peptide analogue, Ac-Phe-Phe-NH2, self-assembled into similar tubular structures. We later explored other amine and carboxyl modified diphenylalanine peptide analogues and revealed that these dipeptides can form ordered tubular structures at the nanometric scale. Moreover, a very similar peptide, the diphenylglycine, self-assembled into ordered nano-spherical assemblies. Here we extend our research and explore the self-assembly of other homo-aromatic dipeptides in which their phenyl side-chains are modified with halogen atoms (di-para-fluoro-Phe, di-pentafluoro-Phe, di-para-iodo-Phe), additional phenyl groups (di-4-phenyl-Phe), or with nitro substitutions (di-para-nitro-Phe). We also probed the effect of the alteration of the phenyl groups with naphtyl groups (di-D-1-Nal and di-D-2-Nal). In all cases, well-ordered nanostructures were obtained and studied by scanning electron microscopy, transmission electron microscopy and vibrational spectroscopy. Taken together, the current work and previous ones define the homo-aromatic dipeptide as a central motif for the formation of ordered self-assembled tubular, spherical and two-dimensional structures at the nano-scale.

Amino Acid Sequence↗
PubMed2006

Thermal and chemical stability of diphenylalanine peptide nanotubes: implications for nanotechnological applications.

The diphenylalanine peptide, the core recognition motif of the beta-amyloid polypeptide, efficiently self-assembles into discrete, well-ordered nanotubes. Here, we describe the notable thermal and chemical stability of these tubular structures both in aqueous solution and under dry conditions. Scanning and transmission electron microscopy (SEM and TEM) as well as atomic force microscopy (AFM) revealed the stability of the nanotubes in aqueous solution at temperatures above the boiling point of water upon autoclave treatment. The nanotubes preserved their secondary structure at temperatures up to 90 degrees C, as shown by circular dichroism (CD) spectra. Cold field emission gun (CFEG) high-resolution scanning electron microscope (HRSEM) and thermogravimetric analysis (TGA) of the peptide nanotubes after dry heat revealed durability at higher temperature. It was shown that the thermal stability of diphenylalanine peptide nanotubes is significantly higher than that of a nonassembling dipeptide, dialanine. In addition to thermal stability, the peptide nanotubes were chemically stable in organic solvents such as ethanol, methanol, 2-propanol, acetone, and acetonitrile, as shown by SEM analysis. Moreover, the acetone environment enabled AFM imaging of the nanotubes in solution. The significant thermal and chemical stability of the peptide nanotubes demonstrated here points toward their possible use in conventional microelectronic and microelectromechanics processes and fabrication into functional nanotechnological devices.

Dipeptides↗
PubMed2006

Fabrication of coaxial metal nanocables using a self-assembled peptide nanotube scaffold.

The design and fabrication of complex nanostructures with specific geometry and composition is one of the main challenges of nanotechnology. Here we demonstrate the devise of metal-insulator-metal, trilayered, coaxial nanocables. Such coaxial geometry may give rise to useful and unique electromagnetic properties. We have fabricated these nanostructures using a scaffold of self-assembled peptide nanotubes. Gold nanoparticles were bound to the surface of peptide nanotubes via a common molecular recognition element that was included in various linker peptides. This enabled us to promote site-specific metal reduction and to create the coaxial nanostructure. Using electron microscopy, 1H NMR spectra, and energy-dispersive X-ray analysis, we monitored the different steps within the process, gaining further understanding of its mechanism.

Crystallization↗
PubMed2006

The early stages of amyloid formation: biophysical and structural characterization of human calcitonin pre-fibrillar assemblies.

Amyloid fibril formation is a nucleation dependent process characterized by a lag-phase prior to the appearance of detectable amyloid fibrils. While the three-dimensional structure of amyloid fibrils at atomic resolution is just beginning to be elucidated, the early process of monomers assembly into oligomers is less understood. Understanding the dynamic processes that lead to the formation of these intermediates is highly important as these assemblies might be the most pathological ones. Here, we investigated the biophysical and structural features characterizing the early stage assemblies formed by the human hormone calcitonin. We calculated the initial nucleus size by experimentally determining the dependence between the lag-time length and the hCT concentrations. We used size exclusion chromatography and dynamic light scattering in order to characterize the dynamic growth process of preliminary intermediates transformed into larger structures. The early structures were visualized using high-resolution transmission electron microscopy. Annular pore-like structures were observed along with protofibrilar structures. This observed morphology is similar to structures revealed during the fibrillization processes of beta-amyloid, alpha-synuclein, and islet amyloid polypeptide, suggesting that these intermediates represent a generic early structure conformation. The results introduced here imply that a variety of intermediate assemblies are formed during the early stages of amyloid fibril formation. The characterizing of their structural features and assembly kinetics will contribute to the rational design of inhibitors directed towards early structure assemblies.

Amyloid↗
PubMed2006

The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain.

Several proteins that interact with cell surface glycolipids share a common fold with a solvent-exposed aromatic residue that stacks onto a sugar ring of the glycolipid (CH-pi stacking interaction). Stacking interactions between aromatic residues (pi-pi stacking) also play a pivotal role in the assembly process, including many cases of amyloid fibril formation. We found a structural similarity between a typical glycolipid-binding domain (the V3 loop of HIV-1 gp120) and the minimal amyloid-forming fragment of the human islet amyloid polypeptide, i.e. the octapeptide core module NFGAILSS. In a monolayer assay at the air-water interface, the NFGAILSS peptide specifically interacted with the glycolipid lactosylceramide. The interaction appears to require an aromatic residue, as NLGAILSS was poorly recognized by lactosylceramide, whereas NYGAILSS behaved like NFGAILSS. In addition, we observed that the full-length human islet amyloid polypeptide (1-37) did interact with a monolayer of lactosylceramide, and that the glycolipid film significantly affected the aggregation process of the peptide. As glycolipid-V3 interactions are efficiently inhibited by suramin, a polyaromatic compound, we investigated the effects of suramin on amyloid formation by human islet amyloid polypeptide. We found that suramin inhibited amyloid fibril formation at low concentrations, but dramatically stimulated the process at high concentrations. Taken together, our results indicate that the minimal amyloid-forming fragment of human islet amyloid polypeptide is a glycolipid-binding domain, and provide further experimental support for the role of aromatic pi-pi and CH-pi stacking interactions in the molecular control of the amyloidogenesis process.

Amyloid↗
PubMed2006

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 Alzheimer's disease, Parkinson's 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.

Amyloid beta-Peptides↗
PubMed2005

The preferred conformation of the tripeptide Ala-Phe-Ala in water is an inverse gamma-turn: implications for protein folding and drug design.

Recent studies have provided evidence that peptides as short as tripeptides do adopt preferred conformations. Here we report that the tripeptide Ala-Phe-Ala (AFA) in aqueous solution preferentially forms an inverse gamma-turn. Circular dichroism (CD) indicated the presence of a predominant turn structure, and Fourier transform infrared (FTIR) bands suggested the presence of a gamma-turn forming a bifurcated H-bond with the solvent molecules. The high-resolution structure was obtained by a combined use of NMR spectroscopy and calculations. On the basis of 30 unambiguous ROESY-derived distance restraints (including the Halpha-NH NOE between Ala(1) and Ala(3) and a hydrogen bond between the CO group of Ala(1) and the NH group of Ala(3)), calculations clearly demonstrated the presence of an inverse gamma-turn centered on Phe(2). From NOE data, we estimated a mole fraction for the gamma-turn of 0.65. Since for AFA an extended beta-strand was also reported [Eker, F., Griebenow, K., Cao, X., Nafie, L. A., and Schweitzer-Stenner, R. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 10054-10059], we investigated the possibility that gamma-turn and beta-strand may represent two major conformations. By using a best-fit procedure that calculated experimental NOEs as weighted averages of the effects originating from both structures, we were able to calculate with good accuracy the backbone NOEs at 280 K in terms of the two limiting conformers, yielding a mole fraction for the gamma-turn and beta-strand conformations of 0.60 and 0.40, respectively, in good agreement with those found by NOE data. The implication of the existence of a preferred conformation by a small structural element is discussed in the context of the nucleation of protein folding events and the design of small peptide and peptidomimetic drugs.

Alanine↗
PubMed2005

The formation of Escherichia coli curli amyloid fibrils is mediated by prion-like peptide repeats.

Amyloid fibril formation is the hallmark of major human maladies including Alzheimer's disease, type II diabetes, and prion diseases. Prion-like phenomena were also observed in yeast. Although not evolutionarily related, one similarity between the animal PrP and the yeast Sup35 prion proteins is the occurrence of short peptide repeats that are assumed to play a key role in the assembly of the amyloid structures. It was recently demonstrated that typical amyloid fibril formation is associated with biofilm formation by Escherichia coli. Here, we note the functional and structural similarity between oligopeptide repeats of the major curli protein and those of animal and yeast prions. We demonstrate that synthetic peptides corresponding to the repeats form fibrillar structures. Furthermore, conjugation of beta-breaker elements to the prion-like repeat significantly inhibits amyloid formation and cell invasion of curli-expressing bacteria. This implies a functional role of the repeat in the self-assembly of the fibrils. Since mammal prion, yeast prion, and curli protein are evolutionarily distinct, the conserved peptide repeats most likely define an optimized self-association motif that was independently evolved by diverse systems.

Amino Acid Sequence↗
PubMed2005

Peptide nanotube-modified electrodes for enzyme-biosensor applications.

The fabrication and notably improved performance of composite electrodes based on modified self-assembled diphenylalanine peptide nanotubes is described. Peptide nanotubes were attached to gold electrodes, and we studied the resulting electrochemical behavior using cyclic voltammetry and chronoamperometry. The peptide nanotube-based electrodes demonstrated a direct and unmediated response to hydrogen peroxide and NADH at a potential of +0.4 V (vs SCE). This biosensor enables a sensitive determination of glucose by monitoring the hydrogen peroxide produced by an enzymatic reaction between the glucose oxidase attached to the peptide nanotubes and glucose. In addition, the marked electrocatalytic activity toward NADH enabled a sensitive detection of ethanol using ethanol dehydrogenase and NAD+. The peptide nanotube-based amperometric biosensor provides a potential new tool for sensitive biosensors and biomolecular diagnostics.

Biosensing Techniques↗
PubMed2005

The YoeB toxin is a folded protein that forms a physical complex with the unfolded YefM antitoxin. Implications for a structural-based differential stability of toxin-antitoxin systems.

The chromosomal YoeB-YefM toxin-antitoxin module common to numerous strains of bacteria is presumed to have a significant role in survival under stringent conditions. Recently we showed that the purified YefM antitoxin is a natively unfolded protein, as we previously reported for the Phd antitoxin in the P1 phage Doc-Phd toxin-antitoxin system. Here we report the purification and structural properties of the YoeB toxin and present physical evidence for the existence of a tight YoeB. YefM polypeptide complex in solution. YoeB and YefM proteins co-eluted as single peaks in sequential Ni-affinity FPLC and Q-Sepharose ion-exchange chromatography implying the formation of a YoeB. YefM complex. The unstable antitoxin was removed from the mixture by natural proteolysis, and the residual YoeB protein was purified using ion exchange chromatography. Fluorescence anisotropy studies of the purified YoeB and YefM proteins showed a 2:1 stoichiometry of the complex, providing direct evidence for a physical complex between the proteins. Near- and far-UV circular dichroism spectroscopy of the purified toxin revealed that, similar to the Doc toxin, YoeB is a well-folded protein. Thermal denaturation experiments confirmed the conformational stability of the YoeB toxin, which underwent reversible thermal unfolding at temperatures up to 56 degrees C. The thermodynamic features of the toxin-antitoxin complex were similar. Taken together, our results support the notion of a correlation between differential physiological and structural stability in toxin-antitoxin modules.

Anisotropy↗
PubMed2005

Energy landscape of amyloidogenic peptide oligomerization by parallel-tempering molecular dynamics simulation: significant role of Asn ladder.

Recent evidence suggests that amyloidogenic oligomers may be the toxic species in cell cultures. Thus, it is crucial to understand their structure and oligomerization mechanism in atomistic detail. By employing tens of fast central processing units and an advanced phase-space sampling algorithm, parallel-tempering molecular dynamics, we have explored the energy landscape of amyloidogenic peptide oligomerization in explicit water. A pentapeptide, DFNKF, derived from human calcitonin and its mutant, DFAKF, was simulated with a total simulation time of approximately 500 ns. The detailed oligomerization process of a DFNKF parallel beta-sheet formation at 300 K has been characterized. The assembly of a parallel beta-sheet from the amorphous state mainly occurs via a "bottleneck" channel where the interstrand Asn-Asn stacking is the major interaction. The interactions of Asn-Asn stacking include both backbone and side-chain hydrogen bonds. The Asn-Asn interactions work like "glue" by sticking the DFNKF strands together and assist the "on-pathway" oligomerization. The Asn-Asn stacking observed here is similar to the Asn ladder commonly found in globular beta-helix proteins. A control run shows that when Asn is mutated to Ala, the stability and population of the DFAKF parallel beta-sheet is decreased. Furthermore, our in vitro mutagenesis experiments show that the ability of DFAKF peptides to form amyloid fibrils is significantly reduced, in agreement with the simulations. Knowledge of the energy landscape of oligomerization may provide hints for rational drug design, preventing amyloid-associated diseases.

Algorithms↗