Β-sheet Peptide Research Articles

Controlling the Self-Assembly and Material Properties of β-Sheet Peptide Hydrogels by Modulating Intermolecular Interactions.

Self-assembling peptides are a promising biomaterial with potential applications in medical devices and drug delivery. In the right combination of conditions, self-assembling peptides can form self-supporting hydrogels. Here, we describe how balancing attractive and repulsive intermolecular forces is critical for successful hydrogel formation. Electrostatic repulsion is tuned by altering the peptide's net charge, and intermolecular attractions are controlled through the degree of hydrogen bonding between specific amino acid residues. We find that an overall net peptide charge of +/-2 is optimal to facilitate the assembly of self-supporting hydrogels. If the net peptide charge is too low then dense aggregates form, while a high molecular charge inhibits the formation of larger structures. At a constant charge, altering the terminal amino acids from glutamine to serine decreases the degree of hydrogen bonding within the assembling network. This tunes the viscoelastic properties of the gel, reducing the elastic modulus by two to three orders of magnitude. Finally, hydrogels could be formed from glutamine-rich, highly charged peptides by mixing the peptides in combinations with a resultant net charge of +/-2. These results illustrate how understanding and controlling self-assembly mechanisms through modulating intermolecular interactions can be exploited to derive a range of structures with tuneable properties.

Controlled Drug Release from Hydrogel Formulations for The Localised Delivery of Anticancer Agents to Solid Tumors

The hydrogel of the β-sheet self-assembled peptides is one of the powerful vehicles for the drug delivery and other biomedical applications. This class of hydrogel contains both hydrophilic and hydrophobic moieties. Therefore, it plays an integral part in the delivering of the hydrophobic drugs, which considers as a main challenge to overcome when dealing with hydrogels, this is because hydrogels are hydrophilic in nature. Herein, Doxorubicin has been used as a model anticancer agent because it is the most widely known as an anthracycline antibiotic with high anticancer activity. The major challenge with this chemotherapeutic agent its poor aqueous solubility, thus attempts have been made to transform it into hydrogel via hydrophobic interactions. The release of doxorubicin from the hydrogels at the tumour cells, is the vital aim here. Controlling the Dox release has been achievable through monitoring several parameters, such as the gel concentrations, PH, time, and the number of lysine residues. The mechanical properties, secondary structure and the morphology of the peptide hydrogels and Dox hydrogels were also assessed, via using the Rheometer, FTIR and SEM.

Conformationally Restricted β-Sheet Breaker Peptides Incorporating Cyclic α-Methylisoserine Sulfamidates.

Peptides containing variations of the β-amyloid hydrophobic core and five-membered sulfamidates derived from β-amino acid α-methylisoserine have been synthesized and fully characterized in the gas phase, solid state and in aqueous solution by a combination of experimental and computational techniques. The cyclic sulfamidate group effectively locks the secondary structure at the N-terminus of such hybrid peptides imposing a conformational restriction and stabilizing non-extended structures. This conformational bias, which is maintained in the gas phase, solid state and aqueous solution, is shown to be resistant to structure templating through assays of in vitro β-amyloid aggregation, acting as β-sheet breaker peptides with moderate activity.

An Engineered β-Hairpin Peptide Forming Thermostable Complexes with ZnII , NiII , and CuII through a His3 Site.

The three-dimensional structure of a peptide, which determines its function, can denature at elevated temperatures, in the presence of chaotropic reagents, or in organic solvents. These factors limit the applicability of peptides. Herein, we present an engineered β-hairpin peptide containing a His3 site that forms complexes with ZnII , NiII , and CuII . Circular dichroism spectroscopy shows that the peptide-metal complexes exhibit melting temperatures up to 80 °C and remain folded in 6 M guanidine hydrochloride as well as in organic solvents. Intrinsic fluorescence titration experiments were used to determine the dissociation constants of metal binding in the nano- to sub-nanomolar range. The coordination geometry of the peptide-CuII complex was studied by EPR spectroscopy, and a distorted square planar coordination geometry with weak interactions to axial ligands was revealed. Due to their impressive stability, the presented peptide-metal complexes open up interesting fields of application, such as the development of a new class of peptide-metal catalysts for stereoselective organic synthesis or the directed design of extremophilic β-sheet peptides.

10-Residue MyD88-Peptide Adopts β-Sheet Structure, Self-Assembles, Binds to Lipopolysaccharides, and Rescues Mice from Endotoxin-Mediated Lung-Infection and Death.

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine-valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

Expanding the Landscape of Amino Acid-Rich Antimicrobial Peptides: Definition, Deployment in Nature, Implications for Peptide Design and Therapeutic Potential.

Unlike the α-helical and β-sheet antimicrobial peptides (AMPs), our knowledge on amino acid-rich AMPs is limited. This article conducts a systematic study of rich AMPs (>25%) from different life kingdoms based on the Antimicrobial Peptide Database (APD) using the program R. Of 3425 peptides, 724 rich AMPs were identified. Rich AMPs are more common in animals and bacteria than in plants. In different animal classes, a unique set of rich AMPs is deployed. While histidine, proline, and arginine-rich AMPs are abundant in mammals, alanine, glycine, and leucine-rich AMPs are common in amphibians. Ten amino acids (Ala, Cys, Gly, His, Ile, Lys, Leu, Pro, Arg, and Val) are frequently observed in rich AMPs, seven (Asp, Glu, Phe, Ser, Thr, Trp, and Tyr) are occasionally observed, and three (Met, Asn, and Gln) were not yet found. Leucine is much more frequent in forming rich AMPs than either valine or isoleucine. To date, no natural AMPs are simultaneously rich in leucine and lysine, while proline, tryptophan, and cysteine-rich peptides can simultaneously be rich in arginine. These findings can be utilized to guide peptide design. Since multiple candidates are potent against antibiotic-resistant bacteria, rich AMPs stand out as promising future antibiotics.

Synthetic Amphipathic β-Sheet Temporin-Derived Peptide with Dual Antibacterial and Anti-Inflammatory Activities.

Temporin family is one of the largest among antimicrobial peptides (AMPs), which act mainly by penetrating and disrupting the bacterial membranes. To further understand the relationship between the physical-chemical properties and their antimicrobial activity and selectivity, an analogue of Temporin L, [Nle1, dLeu9, dLys10]TL (Nle-Phe-Val-Pro-Trp-Phe-Lys-Phe-dLeu-dLys-Arg-Ile-Leu-CONH2) has been developed in the present work. The design strategy consisted of the addition of a norleucine residue at the N-terminus of the lead peptide sequence, [dLeu9, dLys10]TL, previously developed by our group. This modification promoted an increase of peptide hydrophobicity and, interestingly, more efficient activity against both Gram-positive and Gram-negative strains, without affecting human keratinocytes and red blood cells survival compared to the lead peptide. Thus, this novel compound was subjected to biophysical studies, which showed that the peptide [Nle1, dLeu9, dLys10]TL is unstructured in water, while it adopts β-type conformation in liposomes mimicking bacterial membranes, in contrast to its lead peptide forming α-helical aggregates. After its aggregation in the bacterial membrane, [Nle1, dLeu9, dLys10]TL induced membrane destabilization and deformation. In addition, the increase of peptide hydrophobicity did not cause a loss of anti-inflammatory activity of the peptide [Nle1, dLeu9, dLys10]TL in comparison with its lead peptide. In this study, our results demonstrated that positive net charge, optimum hydrophobic−hydrophilic balance, and chain length remain the most important parameters to be addressed while designing small cationic AMPs.

Inhibiting UCH-L5: Rational Design of a Cyclic Ubiquitin-Based Peptide Inhibitor.

The ubiquitin-proteasome system is an essential regulator of many cellular processes including controlling protein homeostasis. The degradation of proteins by the multi-subunit proteasome complex is tightly regulated through a series of checkpoints, amongst which are a set of deubiquitinating proteases (DUBs). The proteasome-associated DUBs, UCH-L5 (Ubiquitin carboxyl-terminal hydrolase isozyme L5) and USP14 (Ubiquitin-specific protease 14), and the integral-DUB in the proteasome, Rpn11, is known to regulate proteasomal degradation by deubiquitination of distinct substrates. Although selective inhibitors for USP14 and Rpn11 have been recently developed, there are no known inhibitors that selectively bind to UCH-L5. The X-ray structure of the Ubiquitin (Ub) bound to UCH-L5 shows a β-sheet hairpin in Ub that contains a crucial hydrophobic patch involved in the interaction with UCH-L5. Herein, we designed and developed both a Ub sequence-based linear- and cyclic- β-sheet hairpin peptide that was found to preferably inhibit UCH-L5. We show that these peptides have low micromolar IC50 values and the cyclic peptide competes with the activity-based UbVME (Ubiquitin-Vinyl-Methyl-Ester) probe for UCH-L5, binding in a concentration-dependent manner. We further establish the selectivity profile of the cyclic peptide for UCH-L5 compared to other members of the UCH-DUB family and other cysteine DUBs in cell lysate. Furthermore, the cyclic peptide infiltrated cells resulting in the accumulation of polyUb chains, and was found to be non-toxic at the concentrations used here. Taken together, our data suggest that the cyclic peptide permeates the cell membrane, inhibits UCH-L5 by possibly blocking its deubiquitinating function, and contributes to the accumulation of polyubiquitinated substrates. The implications of inhibiting UCH-L5 in the context of the 26S proteasome render it an attractive candidate for further development as a potential selective inhibitor for therapeutic purposes.

Promoting effect of a calcium-responsive self-assembly β-sheet peptide on collagen intrafibrillar mineralization.

Recently, a de novo synthetic calcium-responsive self-assembly β-sheet peptide ID8 (Ile-Asp-Ile-Asp-Ile-Asp-Ile-Asp) has been developed to serve as the template inducing hydroxyapatite nucleation. The aim of this study was to evaluate the effect of ID8 on intrafibrillar mineralization of collagen making full use of its self-assembly ability. The mineralization experiments were carried out in vitro on both bare Type I collagen and fully demineralized dentin samples. The calcium-responsive self-assembly of ID8 was revealed by circular dichroism spectrum, 8-anilino-1-naphthalenesulfonic acid ammonium salt hydrate assay, attenuated total reflection Fourier transform infrared spectrum (ATR-FTIR) and transmission electron microscope (TEM). Polyacrylic acid (450 kDa) with a concentration of 100 μg ml−1 was selected as the nucleation inhibitor based on the determination of turbidimetry and TEM with selected area electron diffraction (TEM-SAED). The results showed that collagen intrafibrillar mineralization was significantly promoted with the pretreatment of self-assembly ID8 detected by TEM-SAED, SEM, X-ray diffraction and ATR-FTIR. The pretreatment of collagen utilizing self-assembly ID8 not only enhanced intermolecular hydrogen bonding but also contributed to calcium retention inside collagen and significantly increased the hydrophilicity of collagen. These results indicated that peptides with self-assembly properties like ID8 are expected to be potential tools for biomimetic mineralization of collagen.

Cationic peptides template the assembly of polyoxometalates into ultrathin nanosheets with in-plane ordered arrangements.

Ultrathin polyoxometalate nanosheets with an in-plane alignment have been constructed in aqueous solution with the assistance of cationic peptides. Different POMs varying in topology, size, and charges could be templated into ordered 2D nanostructures based on this generalized approach, allowing the fabrication of customizable 2D materials.

Enantiomeric β-sheet peptides from Aβ form homochiral pleated β-sheets rather than heterochiral rippled β-sheets.

In 1953, Pauling and Corey postulated “rippled” β-sheets, composed of a mixture of d- and l-peptide strands, as a hypothetical alternative to the now well-established structures of “pleated” β-sheets, which they proposed as a component of all-l-proteins. Growing interest in rippled β-sheets over the past decade has led to the development of mixtures of d- and l-peptides for biomedical applications, and a theory has emerged that mixtures of enantiomeric β-sheet peptides prefer to co-assemble in a heterochiral fashion to form rippled β-sheets. Intrigued by conflicting reports that enantiomeric β-sheet peptides prefer to self-assemble in a homochiral fashion to form pleated β-sheets, we set out address this controversy using two β-sheet peptides derived from Aβ17–23 and Aβ30–36, peptides 1a and 1b. Each of these peptides self-assembles to form tetramers comprising sandwiches of β-sheet dimers in aqueous solution. Through solution-phase NMR spectroscopy, we characterize the different species formed when peptides 1a and 1b are mixed with their respective d-enantiomers, peptides ent-1a and ent-1b. 1H NMR, DOSY, and 1H,15N-HSQC experiments reveal that mixing peptides 1a and ent-1a results in the predominant formation of homochiral tetramers, with a smaller fraction of a new heterochiral tetramer, and mixing peptides 1b and ent-1b does not result in any detectable heterochiral assembly. 15N-edited NOESY reveals that the heterochiral tetramer formed by peptides 1a and ent-1a is composed of two homochiral dimers. Collectively, these NMR studies of Aβ-derived peptides provide compelling evidence that enantiomeric β-sheet peptides prefer to self-assemble in a homochiral fashion in aqueous solution.

Switchable Zinc(II)-Responsive Globular β-Sheet Peptide.

The natural function of many proteins depends on their ability to switch their conformation driven by environmental changes. In this work, we present a small, monomeric β-sheet peptide that switches between a molten globule and a folded state through Zn(II) binding. The solvent-exposed hydrophobic core on the β-sheet surface was substituted by a His3-site, whereas the internal hydrophobic core was left intact. Zn(II) is specifically recognized by the peptide relative to other divalent metal ions, binds in the lower micromolar range, and can be removed and re-added without denaturation of the peptide. In addition, the peptide is fully pH-switchable, has a pKa of about 6, and survives several cycles of acidification and neutralization. In-depth structural characterization of the switch was achieved by concerted application of circular dichroism (CD) and multinuclear NMR spectroscopy. Thus, this study represents a viable approach toward a globular β-sheet Zn(II) mini-receptor prototype.

Conformational Preferences and Antiproliferative Activity of Peptidomimetics Containing Methyl 1'-Aminoferrocene-1-carboxylate and Turn-Forming Homo- and Heterochiral Pro-Ala Motifs.

The concept of peptidomimetics is based on structural modifications of natural peptides that aim not only to mimic their 3D shape and biological function, but also to reduce their limitations. The peptidomimetic approach is used in medicinal chemistry to develop drug-like compounds that are more active and selective than natural peptides and have fewer side effects. One of the synthetic strategies for obtaining peptidomimetics involves mimicking peptide α-helices, β-sheets or turns. Turns are usually located on the protein surface where they interact with various receptors and are therefore involved in numerous biological events. Among the various synthetic tools for turn mimetic design reported so far, our group uses an approach based on the insertion of different ferrocene templates into the peptide backbone that both induce turn formation and reduce conformational flexibility. Here, we conjugated methyl 1′-aminoferrocene-carboxylate with homo- and heterochiral Pro-Ala dipeptides to investigate the turn formation potential and antiproliferative properties of the resulting peptidomimetics 2–5. Detailed spectroscopic (IR, NMR, CD), X-ray and DFT studies showed that the heterochiral conjugates 2 and 3 were more suitable for the formation of β-turns. Cell viability study, clonogenic assay and cell death analysis showed the highest biological potential of homochiral peptide 4.

Highly Tough, Stretchable, and Enzymatically Degradable Hydrogels Modulated by Bioinspired Hydrophobic β-Sheet Peptides.

Peptide-based supramolecular hydrogels have attracted great attention due to their good biocompatibility and biodegradability and have become promising candidates for biomedical applications. The bottom-up self-assembly endows the peptides with a highly ordered secondary structure, which has proven to be an effective strategy to improve the mechanical properties of hydrogels through strong physical interactions and energy dissipation. Inspired by the excellent mechanical properties of spider-silk, which can be attributed to the rich β-sheet crystal formation by the hydrophobic peptide fragment, a hydrophobic peptide (HP) that can form a β-sheet assembly was designed and introduced into a poly(vinyl alcohol) (PVA) scaffold to improve mechanical properties of hydrogels by the cooperative intermolecular physical interactions. Compared with hydrogels without peptide grafting (P-HP0), the strong β-sheet self-assembly domain endows the hybrid hydrogels (P-HP20, P-HP29, and P-HP37) with high strength and toughness. The fracture tensile strength increased from 0.3 to 2.1 MPa (7 times), the toughness increased from 0.4 to 21.6 MJ m-3 (54 times), and the compressive strength increased from 0.33 to 10.43 MPa (31 times) at 75% strain. Moreover, the hybrid hydrogels are enzymatically degradable due to the dominant contribution of the β-sheet assembly for network cross-linking. Combining the good biocompatibility and sustained drug release of the constructed hydrogels, this hydrophobic β-sheet peptide represents a promising candidate for the rational design of hydrogels for biomedical applications.

Quantitative nanomechanical properties evaluation of a family of β-sheet peptide fibres using rapid bimodal AFM

Self-assembling peptides have become important building blocks for materials design (e.g. hydrogels) and play a crucial role in a range of diseases including Alzheimer and Parkinson. In this context, accessing the nanomechanical properties of ubiquitous β-sheet rich nanofibres (e.g.: amyloids) is key to the formulation of materials and design of therapies. Although the bulk mechanical properties of hydrogels can easily be accessed using common techniques and equipment, the mechanical properties of their constituent fibres, in particular if with radii in the nanometre scale, are more challenging to measure and estimate. In this work we show for the first time how the rapid nanomechanical mapping technique: amplitude modulation-frequency modulation (AM-FM), can be used to determine the heights, Young's moduli and viscosity coefficients of a series of β-sheet peptide nanofibres with high statistical confidence. Our results show how peptide sequence and in particular length, charge and interaction with the substrate affect the viscoelastic properties of the peptide fibres.

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase.

Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic β-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

Catalytically active peptides affected by self-assembly and residues order

Amphiphilic peptides that induce catalysis are interesting alternatives to natural enzymes thanks to robustness of their synthesis and the ability to induce certain types of conformations by specific motifs of amino acid sequences. Various studies aimed at mimicking the activity of serine proteases by designed peptides. Here we demonstrate that the order by which the catalytic triad residues are positioned along amphiphilic β-strands influences both assembly structures and catalytic activity. A set of three β-sheet amphiphilic peptides, decorated with different orders of the catalytic triad amino acids, Glu, His and Ser along the strands were evaluated for their catalytic hydrolysis efficiency of p-nitrophenyl acetate (pNPA) substrate. Among the three peptides, Ac-Cys-Phe-Glu-Phe-Ser-Phe-His-Phe-Pro-NH2 (ESH) achieved the greatest catalytic efficiency with a value of 0.19 M−1 s−1, at peptide concentration of 250 μM. This study sheds light on an overlooked factor in designing catalytic amphiphilic assemblies whereby charged residues that make up the active sites, are in fact engaged in intermolecular stabilizing interactions that in turn may hamper their catalytic action.

Gold meets peptides in a hybrid coil.

Twisted structures are assembled cooperatively by β-sheet peptides and gold nanorods

Enhancing the binding of the β-sheet breaker peptide LPFFD to the amyloid-β fibrils by aromatic modifications: A molecular dynamics simulation study

Alzheimer’s is a fatal neurodegenerative disease for which there is no cure at present. The disease is characterized by the presence of plaques in the brains of a patient, which are composed mainly of aggregates of the amyloid-β peptide in the form of β-sheet fibrils. Here, we investigated the possibility of exploiting the superior binding ability of aromatic amino acids to a particular model of the amyloid-β fibrils. which is a difficult target for drug design. The β-sheet breaker peptide LPFFD was modified with aromatic amino acids and its binding to these fibrils was studied. We found that the orientation and the electrostatic complementarity of the modified peptide with respect to the fibrils played a crucial role in determining whether its binding was improved by the aromatic amino acids. The modified LPFFD peptides were able to bind to those fibril residues. which are important in the aggregation of amyloid-β peptides and thus can potentially inhibit the further aggregation of the amyloid-beta peptides by blocking their interactions. We found that the tryptophan modified LPFFD peptides had the best binding affinities. In most cases, the aromatic amino acids in the N-terminus of the modified peptides made more contacts with the fibrils than those in the C-terminus. We also found that increasing the aromatic content did not significantly improve the binding of the LPFFD peptide to the fibrils. Our study can serve as a basis for the design of novel peptide-based drugs for Alzheimer’s disease in which aromatic interactions play an important role.

De Novo-Designed β-Sheet Heme Proteins.

The field of de novo protein design has met with considerable success over the past few decades. Heme, a cofactor, has often been introduced to impart a diverse array of functions to a protein, ranging from electron transport to respiration. In nature, heme is found to occur predominantly in α-helical structures over β-sheets, which has resulted in significant designs of heme proteins utilizing coiled-coil helices. By contrast, there are only a few known β-sheet proteins that bind heme and designs of β-sheets frequently result in amyloid-like aggregates. This review reflects on our success in designing a series of multistranded β-sheet heme binding peptides that are well folded in both aqueous and membrane-like environments. Initially, we designed a β-hairpin peptide that self-assembles to bind heme and performs peroxidase activity in membrane. The β-hairpin was optimized further to accommodate a heme binding pocket within multistranded β-sheets for catalysis and electron transfer in membranes. Furthermore, we de novo designed and characterized β-sheet peptides and miniproteins that are soluble in an aqueous environment capable of binding single and multiple hemes with high affinity and stability. Collectively, these studies highlight the substantial progress made toward the design of functional β-sheets.