On the design and efficacy assessment of self‐assembling peptide‐based hydrogel‐glycosaminoglycan mixtures for potential repair of early stage cartilage degeneration

Peptide‐based hydrogels are of interest for their potential use in regenerative medicine. Combining these hydrogels with materials that may enhance their physical and biological properties, such as glycosaminoglycans, has the potential to extend their range of biomedical applications, for example in the repair of early cartilage degeneration. The aim of this study was to combine three self‐assembling peptides (P₁₁‐4, P₁₁‐8, and P₁₁‐12) with chondroitin sulphate at two molar ratios of 1:16 and 1:64 in 130 and 230 mM Na⁺ salt concentrations. The study investigates the effects of mixing self‐assembling peptide and glycosaminoglycan on the physical and mechanical properties at 37°C. Peptide alone, chondroitin sulphate alone, and peptide in combination with chondroitin sulphate were analysed using Fourier transform infrared spectroscopy to determine the β‐sheet percentage, transmission electron microscopy to determine the fibril morphology, and rheology to determine the elastic and viscous modulus of the materials. All of the variables (peptide, salt concentration, and chondroitin sulphate molar ratio) had an effect on the mechanical properties, β‐sheet formation, and fibril morphology of the hydrogels. P₁₁‐4 and P₁₁‐8‐chondroitin sulphate mixtures, at both molar ratios, were shown to have a high β‐sheet percentage, dense entangled fibrillar networks, as well as high mechanical stiffness in both (130 and 230 mM) Na⁺ salt solutions when compared with the P₁₁‐12/chondroitin sulphate mixtures. These peptide/chondroitin sulphate hydrogels show promise for biomedical applications in glycosaminoglycan depleted tissues.     

Molecular mechanism of β-sheet self-organization at water-hydrophobic interfaces

The capacity to form β‐sheet structure and to self‐organize into amyloid aggregates is a property shared by many proteins. Severe neurodegenerative pathologies such as Alzheimer's disease are thought to involve the interaction of amyloidogenic protein oligomers with neuronal membranes. To understand the experimentally observed catalysis of amyloid formation by lipid membranes and other water‐hydrophobic interfaces, we examine the physico‐chemical basis of peptide adsorption and aggregation in a model membrane using atomistic molecular simulations. Blocked octapeptides with simple, repetitive sequences, (Gly‐Ala)₄, and (Gly‐Val)₄, are used as models of β‐sheet‐forming polypeptide chains found in the core of amyloid fibrils. In the presence of an n‐octane phase mimicking the core of lipid membranes, the peptides spontaneously partition at the octane‐water interface. The adsorption of nonpolar sidechains displaces the peptides' conformational equilibrium from a heterogeneous ensemble characterized by a high degree of structural disorder toward a more ordered ensemble favoring β‐hairpins and elongated β‐strands. At the interface, peptides spontaneously aggregate and rapidly evolve β‐sheet structure on a 10 to 100 ns time scale, while aqueous aggregates remain amorphous. Catalysis of β‐sheet formation results from the combination of the hydrophobic effect and of reduced conformational entropy of the polypeptide chain. While the former drives interfacial partition and displaces the conformational equilibrium of monomeric peptides, the planar interface further facilitates β‐sheet organization by increasing peptide concentration and reducing the dimensionality of self‐assembly from three to two. These findings suggest a general mechanism for the formation of β‐sheets on the surface of globular proteins and for amyloid self‐organization at hydrophobic interfaces. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of secondary structure and self‐assembly of amelogenin by variable temperature circular dichroism and isothermal titration calorimetry

Amelogenin is a proline‐rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self‐assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary–secondary structure and self‐assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full‐length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered β‐structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self‐assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (ΔH_A) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Peptide contour length determines equilibrium secondary structure in protein‐analogous micelles

This work advances bottom‐up design of bioinspired materials built from peptide‐amphiphiles, which are a class of bioconjugates in which a biofunctional peptide is covalently attached to a hydrophobic moiety that drives self‐assembly in aqueous solution. Specifically, this work highlights the importance of peptide contour length in determining the equilibrium secondary structure of the peptide as well as the self‐assembled (i.e., micelle) geometry. Peptides used here repeat a seven‐amino acid sequence between one and four times to vary peptide contour length while maintaining similar peptide‐peptide interactions. Without a hydrophobic tail, these peptides all exhibit a combination of random coil and α‐helical structure. Upon self‐assembly in the crowded environment of a micellar corona, however, short peptides are prone to β‐sheet structure and cylindrical micelle geometry while longer peptides remain helical in spheroidal micelles. The transition to β‐sheets in short peptides is rapid, whereby amphiphiles first self‐assemble with α‐helical peptide structure, then transition to their equilibrium β‐sheet structure at a rate that depends on both temperature and ionic strength. These results identify peptide contour length as an important control over equilibrium peptide secondary structure and micelle geometry. Furthermore, the time‐dependent nature of the helix‐to‐sheet transition opens the door for shape‐changing bioinspired materials with tunable conversion rates. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 573–581, 2013.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Refolding of Npro fusion proteins

The autoprotease N^pro significantly enhances expression of fused peptides and proteins and drives the formation of inclusion bodies during protein expression. Upon refolding, the autoprotease becomes active and cleaves itself specifically at its own C‐terminus releasing the target protein with its authentic N‐terminus. N^pro wild‐type and its mutant EDDIE, respectively, were fused N‐terminally to the model proteins green fluorescent protein, staphylococcus Protein A domain D, inhibitory peptide of senescence‐evasion‐factor, and the short 16 amino acid peptide pep6His. In comparison with the N^pro wild‐type, the tailored mutant EDDIE displayed an increased rate constant for refolding and cleavage from 1.3 × 10^?4 s^?1 to 3.5 × 10^?4 s^?1, and allowed a 15‐fold higher protein concentration of 1.1 mg/mL when studying pep6His as a fusion partner. For green fluorescent protein, the rate constant was increased from 2.4 × 10^?5 s^?1 to 1.1 × 10^?4 s^?1 when fused to EDDIE. When fused to small target peptides, refolding and cleavage yields were independent of initial protein concentration, even at high concentrations of 3.9 mg/mL, although cleavage rates were strongly influenced by the fusion partner. This behavior differed from conventional 1st order refolding kinetics, where yield strongly depends on initial protein concentration due to an aggregation reaction of higher order. Refolding and cleavage of EDDIE fusion proteins follow a monomolecular reaction for the autoproteolytic cleavage over a wide concentration range. At high protein concentrations, deviations from the model assumptions were observed and thus smaller rate constants were required to approximate the data. Biotechnol. Bioeng. 2009; 104: 774–784 © 2009 Wiley Periodicals, Inc.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Biodegradable delivery system containing a peptide inhibitor of polyglutamine aggregation: a step toward therapeutic development in Huntington's disease

Huntington's and eight other neurodegenerative diseases occur because of CAG repeat expansion mutation culminating into an expanded polyglutamine tract in respective protein. In Huntington's disease (HD), a number of CAG repeats beyond normal repeat length (>36) lead to the formation of mutant protein, the proteolytic cleavage of which induces aggregation in polyglutamine length‐dependent manner. The neurodegeneration in this disease is linked to aggregation, and its inhibition is a potential approach for therapeutic development. Although peptides and other molecules have been developed for inhibiting aggregation, peptides in general are susceptible to degradation in vivo conditions. To understand their clinical significance, they also need to be delivered through blood–brain barrier. Here, for the first time, we have synthesized poly‐d ,l ‐lactide‐co‐glycolide nanoparticles containing a polyglutamine aggregation inhibitor peptide PGQ₉[P²], by nanoprecipitation method. This process yielded less than 200 nm spherical nanoparticles with uniform distribution. Characterization studies by infrared spectroscopy‐based and HPLC‐based assays show the presence of PGQ₉[P²] in nanoparticles. In vitro release kinetics demonstrates that nanoparticles release PGQ₉[P²] by erosion and diffusion processes. When the PGQ₉[P²]‐loaded nanoparticles are incubated with aggregation‐prone Q₃₅P₁₀ peptide, representing N‐terminal part of Huntingtin protein, it arrests the elongation phase of Q₃₅P₁₀ aggregation. These findings propose the first step toward delivery of a peptide inhibitor against polyglutamine aggregation in HD. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.     

A general strategy for the bacterial expression of amyloidogenic peptides using BCL‐XL‐1/2 fusions

Biophysical studies on amyloidogenic and aggregation‐prone peptides often require large quantities of material. However, solid‐phase synthesis, handling, and purification of peptides often present challenges on these scales. Recombinant expression is an attractive alternative because of its low cost, the ability to isotopically label the peptides, and access to sequences exceeding ～50 residues. However, expression systems that seek to solubilize amyloidogenic peptides suffer from low yields, difficult optimizations, and isolation challenges. We present a general strategy for expressing and isolating amyloidogenic peptides in Escherichia coli by fusion to a polypeptide that drives the expression of attached peptides into bacterial inclusion bodies. This scheme minimizes toxicity during bacterial growth and enables the processing and handling of the peptides in denaturing solutions. Immobilized metal affinity chromatography, reverse phase HPLC, and cyanogen bromide cleavage are used to isolate the peptide, followed by further reverse phase HPLC, which yields milligram quantities of the purified peptide. We demonstrate that driving the peptides into inclusion bodies using fusion to BCL‐XL‐1/2 is a general strategy for their expression and isolation, as exemplified by the production of 11 peptides species.     

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti‐diabetic agents

Temporin A (FLPLIGRVLSGIL‐NH₂), temporin F (FLPLIGKVLSGIL‐NH₂), and temporin G (FFPVIGRILNGIL‐NH₂), first identified in skin secretions of the frog Rana temporaria, produced concentration‐dependent stimulation of insulin release from BRIN‐BD11 rat clonal β‐cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca²⁺ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration‐dependent stimulation of insulin release from 1.1B4 human‐derived pancreatic β‐cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN‐BD11 cells against cytokine‐induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon‐like peptide‐1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.     

Production of an anti‐Candida peptide via fed batch and ion exchange chromatography

Interest in peptides as diagnostic and therapeutic materials require their manufacture via either a recombinant or synthetic route. This study examined the former, where a recombinant fusion consisting of an antifungal peptide was expressed and isolated from Escherichia coli. Fed batch fermentation with E. coli harboring an arabinose‐inducible plasmid produced the 12 residue anti‐Candida peptide fused to the N‐terminal of Green Fluorescent Protein (GFP_UV). The purification of the fusion protein, using ion‐exchange chromatography, was monitored by using the intrinsic fluorescence of GFP_UV. The recombinant antifungal peptide was successfully released by cyanogen bromide‐induced cleavage of the fusion protein. The recombinant peptide showed the expected antifungal activity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:865–871, 2016     

Controlling network topology and mechanical properties of co‐assembling peptide hydrogels

Oligopeptides are well‐known to self‐assemble into a wide array of nanostructures including β‐sheet‐rich fibers that when present above a critical concentration become entangled and form self‐supporting hydrogels. The length, quantity, and interactions between fibers influence the mechanical properties of the hydrogel formed and this is typically achieved by varying the peptide concentration, pH, ionic strength, or the addition of a second species or chemical cross‐linking agent. Here, we outline an alternative, facile route to control the mechanical properties of the self‐assembling octa‐peptide, FEFEFKFK (FEKII); simply doping with controlled quantities of its double length peptide, FEFEFKFK‐GG‐FKFKFEFE (FEKII18). The structure and properties of a series of samples were studied here (0–100 M% of FEKII18) using Fourier transform infrared, small angle X‐ray scattering, transmission electron microscopy, and oscillatory rheology. All samples were found to contain elongated, flexible fibers and all mixed samples contained Y‐shaped branch points and parallel fibers which is attributed to the longer peptide self‐assembling within two fibers, thus creating a cross‐link in the network structure. Such behavior was reflected in an increase in the elasticity of the mixed samples with increasing quantity of double peptide. Interestingly the elastic modulus increased up to 30 times the pure FEKII value simply by adding 28 M% of FEKII18. These observations provide an easy, off‐the‐shelf method for an end‐user to control the cross‐linked network structure of the peptide hydrogel, and consequently strength of the hydrogel simply by physically mixing pre‐determined quantities of two similar peptide molecules. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 669–680, 2014.     

Purification, Refolding of Hybrid hIFNγ-Kringle 5 Expressed in Escherichia coli

The DNA sequence coding for plasminogen kringle 5 (pK5), an inhibitor of angiogenesis, was fused with that coding for interferon gamma and over-produced in the form of inactive inclusion bodies in E. coli. The amount of fusion protein was about 40% of total protein produced. The fusion protein contained in the inclusion bodies was solubilized in 8 m urea and purified by anion-exchange chromatography. We employed the orthogonal experimental design L₁₆(4⁵) (5 factors, 4 levels, 16 experiments) procedure for researching the influence of denaturant, aggregation suppressor l-arginine, NaCl, pH, and glycine on the refolding procedure. Our results suggest that the presence of appropriate l-arginine, NaCl, and denaturant in the refolding buffer inhibits the aggregation of the fusion protein and increases the yield of renatured protein with biological activity. The refolded fusion protein, γIFN/pk5, has in vitro anti-endothelial cell proliferation activity. Received: 24 July 2000 / Accepted: 21 September 2000     

Separation of recombinant human vasoactive intestinal peptide–humanin fusion protein via three different approaches

Both vasoactive intestinal peptide (VIP) and humanin (HN) can provide neuroprotection against β-amyloid toxicity and are believed to be beneficial in the treatment of Alzheimer's disease. For the sake of tandem co-expression of recombinant human VIP and HN(rhVIP-HN) fusion protein, new VIP-HN gene is constructed and cloned into the plasmid pET28a(+) and is expressed in Escherichia coli BL21(DE3). Since the fusion protein was present as inclusion bodies, three different preparation approaches are employed to obtain the hexa-histidine tagged rhVIP-HN (His₆-rhVIP-HN) fusion protein by Ni²⁺ chelating resin chromatography. The comparison of the results obtained from the three approaches reveals that renaturation during the separation process (Approach III) is the most efficient for large-scale preparation of His₆-rhVIP-HN. Ultimately, 46 mg of the target protein was obtained from one gram of the inclusion body with purity up to 90%. Since there is not separated step in renaturation procedure, Approach III could be more time-saving, buffer-saving and easy to operate than the other two approaches. These results can be useful for preparation of rhVIP-HN in large scale.     

Development of short and highly potent self‐assembling elastin‐derived pentapeptide repeats containing aromatic amino acid residues

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self‐assembly of elastin into fibers, and the temperature‐mediated self‐assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio‐applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin‐derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe‐containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X¹P²G³V⁴G⁵) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp‐containing elastin‐derived peptide analog (WPGVG)₃ with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin‐derived peptide analogs. Generally, however, the production of long‐chain synthetic polypeptides in quantities sufficient for commercial use remain cost‐prohibitive. Therefore, the identification of (WPGVG)₃, which is a 15‐mer short peptide consisting simply of five natural amino acids and shows temperature‐dependent self‐assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel β‐sheet in crystals

The crystal structure of a tripeptide Boc‐Leu‐Val‐Ac₁₂c‐OMe ( 1 ) is determined, which incorporates a bulky 1‐aminocyclododecane‐1‐carboxylic acid (Ac₁₂c) side chain. The peptide adopts a semi‐extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C^α,α‐dialkylated residue Ac₁₂c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β‐sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac₁₂c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti‐parallel β‐sheet in crystals, twisted or helical β‐sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β‐sheet assembly in crystals. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.