Charged Peptide Research Articles

Context-Dependent Heterotypic Assemblies of Intrinsically Disordered Peptides.

Despite their critical role in context-dependent interactions for protein functions, intrinsically disordered regions (IDRs) are often overlooked for designing peptide assemblies. Here, we exploit IDRs to enable context-dependent heterotypic assemblies of intrinsically disordered peptides, where "context-dependent" refers to assembly behavior driven by interactions with other molecules. By attaching an aromatic segment to oppositely charged intrinsically disordered peptides, we achieve a nanofiber formation. Although the same-charged peptides cannot self-assemble, oppositely charged peptides form heterotypic nanofibers. Cryo-EM analysis reveals a β-sheet arrangement within the ordered core of these nanofibers, conformational heterogeneity, and a disorder-to-order continuum and shows a high number of hydrogen bonds between tyrosine and lysine ε-amine. Additionally, this work demonstrates a post-assembly morphological change resulting from local conformational flexibility. While equal molar mixtures of the charged intrinsically disordered peptides yield nanofibers, doubling the positively charged peptides after assembly produces bundles of nanofibers. Furthermore, reducing the number of aromatic amino acid residues reduces bundle formation. Demonstrating context-dependent self-assembly of intrinsically disordered peptides and revealing atomistic insights into heterotypic assemblies of intrinsically disordered peptides for the first time, this work illustrates a straightforward approach to enable heterotypic intrinsically disordered peptides to self-assemble for the design of adaptive, multifunctional peptide nanomaterials.

Diacylation of Peptides Enables the Construction of Functional Vesicles for Drug-Carrying Liposomes.

Membrane-forming phospholipids are generated in cells by enzymatic diacylation of non-amphiphilic polar head groups. Analogous non-enzymatic processes may have been relevant at the origin of life and could have practical utility in membrane synthesis. However, aqueous head group diacylation is challenging in the absence of enzymes. The use of charged peptides instead of canonical phospholipid head groups offers advantages with respect to ease of acylation and chemical diversity. Here we demonstrate that native chemical ligation (NCL) enables in situ synthesis of diacylated lipopeptides (D-ALPs), which spontaneously self-assemble into micron-sized vesicles resembling cellular membranes. Diacylation occurs between non-amphiphilic peptides possessing an N-terminal cysteine, and acyl thioesters. Peptide head groups endow unique membrane functions, which is demonstrated by incorporation of an arginine-glycine-aspartic acid (RGD) motif, resulting in vesicle targeting to αvβ3 integrin-overexpressing cancer cells. The biocompatibility and functional group programmability of D-ALPs supports their broad utility as membrane mimetics.

NaCl-Responsive Ultrashort Peptide to Trigger Self-Assembly of TPE-Capped Supramolecular Hydrogelator.

With the advantages of less invasiveness and better shape adaptability, in situ-forming hydrogels are desired biomaterials as scaffolds, drug carriers, and so on. Herein, a negatively charged NaCl-responsive ultrashort peptide sequence (EEH) is reported whose electrostatic repulsion can be reduced through the charge-shielding effect. Under physiological conditions, its AIEgen-capped amphiphile TPE-GEEH of low concentration (1 mg/mL) presents NaCl-triggered morphological transformation from micelle to closely packed fiber with enhanced emission, which can be applied to biosense sodium ion (Na+) with high sensitivity and quick response. At a slightly acidic pH, 10 mg/mL TPE-GEEH undergoes sol-gel transition upon addition of NaCl (100 mM) with improved mechanical properties, which should be useful to develop an in situ-forming hydrogel. Overall, our report provides a simple strategy to construct NaCl-responsive assemblies for potential application in biosensors and drug delivery system.

Peptide-Guided Assembly of Silver Nanoparticles for the Diagnosis of HER2-Positive Breast Cancer.

Peptide-engineered nanoparticles have great potential for biomedical research and application. In this work, we have designed and fabricated an electrochemical biosensor based on peptide-guided assembly of silver nanoparticles (AgNPs), in which a peptide is endowed with dual functions to recognize target and guide assembly of AgNPs. As a proof of concept, the performance of this biosensor is validated by quantifying human epidermal growth factor receptor 2 (HER2) protein. In detail, the end of the HER2-specific binding peptide is grafted with a positively charged peptide, which can guide the orderly assembly of AgNPs, while electrochemical signals can be obtained through phosphine-silver coordination. Using this electrochemical biosensor, HER2 protein can be quantified with high sensitivity and specificity, and the limit of detection can be as low as 0.05 pg/mL. Moreover, the antifouling electrode surface prepared by the modification of a layer of antifouling zwitterionic peptide allows this biosensor to be used for the detection of serum HER2 protein from breast cancer patients, which provides the clear evidence for the distinction of HER2+ breast cancer patients and HER2- breast cancer patients.

Analysis of the effects of differently charged peptides on α-amylase and their interaction mechanisms

Nowadays α-amylase is widely used in various fields. Therefore, in this study, the effects of neutral (T0), negatively charged (T8−) and positively charged (T9+) peptides on α-amylase activity were investigated by means of an applied protein electric field, and spectroscopy and molecular dynamics were employed to investigate this mechanism. It was found that the nature of the charge of the peptides had a strong influence on α-amylase activity, with T8− and T9+ increasing and decreasing α-amylase activity, respectively, whereas T0 had no effect on enzyme activity. Fluorescence spectroscopy and circular dichroism results indicated that the charged peptides changed the conformation of α-amylase. Meanwhile, the molecular dynamics results showed that the charged peptides changed the distribution of the surface charge of α-amylase mainly through electrostatic force, which not only changed the conformation of the enzyme, but also altered the microenvironment of the enzyme active centre, which caused α-amylase to become compact or loose to affect the enzyme activity.

End Group Effects on Anion Binding in Tetraglycine Peptide: A Computational Study.

The importance of anions in various processes has led to a search for molecules that can effectively recognize and interact with these anions. This study explores how the tetraglycine [(Gly)4] peptide in its zwitterionic, neutral, and terminally capped forms acts as a receptor for H2PO4 - and HSO4 - anions within the framework of supramolecular host-guest chemistry. Using molecular dynamics (MD) simulations, we obtained the conformations of the receptor-anion complexes. Density functional theory (DFT), quantifies the complexes' interaction energies in both gas and solvent phases. Proton transfer within the zwitterionic complex with H2PO4 - anion alters peptide charge distribution, affecting its conformation and binding site arrangement, as analysed by quantum mechanics/molecular mechanics (QM/MM) methods. Symmetry-adapted perturbation theory (SAPT) and noncovalent interactions analysis highlight the role of electrostatic interactions in these receptor-anion complexes. It emphasizes the key interactions such as N-H⋅⋅⋅⋅O and O-H⋅⋅⋅O=C between the peptide backbone and anions and elucidates the molecular recognition mechanism driven by crucial noncovalent interactions. The termination of the peptide's end groups modulates anion binding sites from the backbone to the charged N-terminal, resulting in distinct binding sites. Our findings provide insights for designing peptides tailored to function as anion receptors in diverse supramolecular chemistry applications.

Double-crosslinked hydrogels and hydrogel beads formed by garlic protein hydrolysates for bioactive encapsulation and gastrointestinal delivery.

Garlic protein is one of the main components of garlic. It has several beneficial characteristics. This study aimed to characterize a double crosslinked hydrogel formed with alginate, calcium ions (Ca2+), and garlic protein hydrolysates (GPH), and to develop hydrogel beads for targeted delivery of bioactive constituents to the gastrointestinal tract. The results indicated that the degree of GPH hydrolysis was approximately 3% following trypsin treatment. The inner structure of the double crosslinked hydrogel showed a honeycomb pattern, with solid-like gel rheology and improved texture properties at a 4% (w/v) GPH concentration. The GPH-based hydrogel beads demonstrated pH sensitivity, swelling in near-neutral and alkaline environments, and the encapsulated paclitaxel (PTX) exhibited an amorphous phase with preferential release in intestinal conditions. The GPH group also achieved greater drug encapsulation efficiency than a soy protein hydrolysate (SPH) group, and proteomic analysis suggested that lower molecular weight and peptide charge favored the formation of peptide-integrated double crosslinking hydrogels. This work indicated that GPH was helpful and could inspire the development of drug delivery systems involving GPH with the required mechanical strength and target-release properties. © 2024 Society of Chemical Industry.

Generative Adversarial Network-Based Augmentation with Noval 2-step Authentication for Anti-coronavirus Peptide Prediction.

The virus poses a longstanding and enduring danger to various forms of life. Despite the ongoing endeavors to combat viral diseases, there exists a necessity to explore and develop novel therapeutic options. Antiviral peptides are bioactive molecules with a favorable toxicity profile, making them promising alternatives for viral infection treatment. Therefore, this article employed a generative adversarial network for antiviral peptide augmentation and a novel two-step authentication process for augmented synthetic peptides to enhance antiviral activity prediction. Additionally, five widely utilized deep learning models were employed for classification purposes. Initially, a GAN was used to augment the antiviral peptide. In a two-step authentication process, the NCBI-BLAST was utilized to identify the antiviral activity resemblance between the synthetic and real peptide. Subsequently, the hydrophobicity, hydrophilicity, hydroxylic nature, positive charge, and negative charge of synthetic and authentic antiviral peptides were compared before their utilization. Later, to examine the impact of authenticated peptide augmentation in the prediction of antiviral peptides, a comparison is conducted with the outcomes of non-peptide augmented prediction. The study demonstrates that the 1-D convolution neural network with augmented peptide exhibits superior performance compared to other employed classifiers and state-of-the-art models. The network attains a mean classification accuracy of 95.41%, an AUC value of 0.95, and an MCC value of 0.90 on the benchmark antiviral and anti-corona peptides dataset. Thus, the performance of the proposed model indicates its efficacy in predicting the antiviral activity of peptides.

A Matter of Charge: Electrostatically Tuned Coassembly of Amphiphilic Peptides.

Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this work coassemblies of amphiphilic and oppositely charged, anionic and cationic, β-sheet peptides are studied, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides show significantly lower critical coassembly concentration (CCC) values compared to those of the individual pure peptides. Intriguingly, the highest formation of coassembled fibrils is found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into β-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic critical assembly concentration (CAC), are also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic investigation of oppositely charged β-sheet peptides over a range of concentrations, in solutions and in hydrogels. The results may be relevant to the fundamental understanding of such intricate charge-driven assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.

Distinct Effects of SARS-CoV-2 Protein Segments on Structural Stability, Amyloidogenic Potential, and α-Synuclein Aggregation.

Amyloidosis is characterized by the abnormal accumulation of misfolded proteins, called amyloid fibrils, leading to diverse clinical manifestations. Recent studies on the amyloidogenesis of SARS-CoV-2 protein segments have raised concerns on their potential link to post-infection neurodegeneration, however, the mechanisms remain unclear. Herein, we investigated the structure, stability, and amyloidogenic propensity of a nine-residue segment (SK9) of the SARS-CoV-2 envelope protein and their impact on neuronal protein α-synuclein (αSyn) aggregation. Specifically, the amino acid sequence of the SK9 wildtype has been modified from a basic and positively charged peptide (SFYVYSRVK), to a nearly neutral and more hydrophobic peptide (SAAVASAVK, labelled as SK9 var1), and to an acidic and negatively charged peptide (SDAVANAVK, labelled as SK9 var2). Our findings reveal that the SK9 wildtype exhibited a pronounced amyloidogenic propensity due to its disordered and unstable nature, while the SK9 variants possessed more ordered and stable structures preventing the amyloid formation. Significantly, the SK9 wildtype demonstrated distinct effect on αSyn aggregation kinetics and aggregate morphology to facilitate the formation of αSyn aggregates with enhanced resistance against enzymatic degradation. This study highlights the potential of modifying short peptide sequences to fine-tune their properties, providing insights into understanding and regulating viral-induced amyloid aggregations.

Injectable self-assembling peptide hydrogel as a promising vitreous substitute

Vitreoretinal diseases pose significant threats to vision, often requiring vitrectomy and substitution of vitreous humor to restore ocular structure and visual function. However, existing substitutes have limitations that compromise patient outcomes. Supramolecular hydrogels, particularly peptide-based formulations, have emerged as promising alternatives due to their superior optical clarity, biocompatibility, and viscoelasticity. In this study, we designed and evaluated two peptide hydrogels, 3K-OX and 3E-OX, bearing positive and negative charges, respectively, as potential vitreous substitutes. Our in vitro findings revealed that the physicochemical properties of the negatively charged peptide hydrogel, 3E-OX, closely resembled those of the native vitreous body, exhibiting optimal light transmittance, refractive index, molecular permeability, and biocompatibility. Animal studies further confirmed the safety and biocompatibility of 3E-OX as a promising vitreous substitute. Notably, we introduced optical coherence tomography for retinal microvascular detection in non-pigmented rabbits, presenting a novel approach to evaluate the performance of intraocular tamponade materials. This work not only expands the utility of peptide hydrogels but also provides valuable insights into the design of vitreous substitutes.

Designing highly tunable anion responsive Cardin-motif peptide based self-assembled nanostructures for accessing diverse cellular response

Several anions present in the extracellular matrix (ECM) not only have significant physiological functions in ECM but also play an important role in regulating peptide-based self-assembly. Herein, we have employed a non-conventional approach to overcome the limitations of the positively charged Cardin-motif peptide that failed to self-assemble at physiological pH. We used a simple and elegant strategy by employing different anions such as HPO42-, Cl- and I- to mask the overall surface charge of peptide. Interestingly, these anions were utilized to modulate the nanostructure formation and mechanical stiffness of peptide hydrogels owing to their differential interactions with water molecules according to the Hofmeister series. Interestingly, these anions induced hydrogels showed diverse cellular responses on two different cell lines, fibroblast and neuronal, indicating diverse application potential of the new scaffold. Thus, this study emphasizes the importance of anions to regulate the self-assembly of Cardin-motif peptide and this approach can be utilized in developing the ideal biomimetic model of ECM for futuristic applications.

Peptide-Driven Assembly of Magnetic Beads for Potentiometric Sensing of Bacterial Enzyme at a Subcellular Level.

Bacterial enzymes with different subcellular localizations play a critical ecological role in biogeochemical processing. However, precisely quantifying enzymes localized at certain subcellular levels, such as extracellular enzymes, has not yet been fully realized due to the complexity and dynamism of the bacterial outer membrane. Here we present a magneto-controlled potentiometric sensing platform for the specific detection of extracellular enzymatic activity. Alkaline phosphatase (ALP), which is one of the crucial hydrolytic enzymes in the ocean, was selected as the target enzyme. Magnetic beads functionalized with an ALP-responsive self-assembled peptide (GGGGGFFFpYpYEEE, MBs-peptides) prevent negatively charged peptides from entering the bacterial outer membrane, thereby enabling direct potentiometric sensing of extracellular ALP both attached to the bacterial cell surface and released into the surrounding environment. The dephosphorylation-triggered assembly of peptide-coupled magnetic beads can be directly and sensitively measured by using a magneto-controlled sensor. In this study, extracellular ALP activity of Pseudomonas aeruginosa at concentrations ranging from 10 to 1.0 × 105 CFU mL-1 was specifically and sensitively monitored. Moreover, this magneto-controlled potentiometric method enabled a simple and accurate assay of ALP activity across different subcellular localizations.

Polarized ATR-FTIR studies of DPPC/DPPG lipid bilayers doped with PLL

The structural characterization of lipid bilayers is crucial for understanding various biological processes and designing effective systems of drugs. Attenuated Total Reflection Fourier Transformed Infrared (ATR-FTIR) spectroscopy is a powerful technique for probing lipid membrane properties, but conventional methods face challenges in discerning molecular orientation and intermolecular interactions. The polarized ATR-FTIR spectroscopy combined with Principal Component Analysis (PCA) was used to investigate the interactions between positively charged peptide (poly-L-lysine (PLL)) and anionic lipid bilayers (dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol, DPPC/DPPG). This integrated approach offers deeper insight into mutual interactions between proteins and lipids of membrane, providing a comprehensive understanding of structure of both protein and lipid bilayers components. The findings of this study shed light on the orientational order of lipid molecules in DPPC/DPPG membrane structure as well as secondary strictures of PPL peptides. It was shown that the presence of PLL peptide disrupts lipid molecule ordering within the lipid bilayer, primarily through electrostatic and H-bond interactions. The utility of polarized ATR-FTIR spectroscopy supported by PCA calculations in elucidating molecular organizations within complex membrane systems is emphasized.

New targets and designed inhibitors of ASAP Arf-GAPs derived from structural characterization of the ASAP1/440-kD ankyrin-B interaction

ASAP1 and its paralog ASAP2 belong to a PI4,5P2-dependent Arf GTPase-activating protein (Arf-GAP) family capable of modulating membrane and cytoskeletal dynamics. ASAPs regulate cell adhesive structures such as invadosomes and focal adhesions during cell attachment and migration. Malfunctioning of ASAP1 has been implicated in the malignant phenotypes of various cancers. Here, we discovered that the SH3 domain of ASAP1 or ASAP2 specifically binds to a 12-residue, positively charged peptide fragment from the 440 kDa giant ankyrin-B, a neuronal axon specific scaffold protein. The high-resolution structure of the ASAP1-SH3 domain in complex with the gAnkB peptide revealed a non-canonical SH3-ligand binding mode with high affinity and specificity. Structural analysis of the complex readily uncovered a consensus ASAP1-SH3 binding motif, which allowed the discovery of a number of previously unknown binding partners of ASAP1-SH3 including Clasp1/Clasp2, ALS2, β-Pix, DAPK3, PHIP, and Limk1. Fittingly, these newly identified ASAP1 binding partners are primarily key modulators of the cytoskeletons. Finally, we designed a cell-penetrating, highly potent ASAP1 SH3 domain binding peptide with a Kd ∼7 nM as a tool for studying the roles of ASAPs in different cellular processes.

Cooperative β-sheet coassembly controls intermolecular orientation of amphiphilic peptide-polydiacetylene conjugates

In this work, we elucidated the structural organization of stimuli-responsive peptide-polydiacetylene (PDA) conjugates that can self-assemble as 1D nanostructures under neutral aqueous conditions. The amino acid sequences bear positively or negatively charged domains at the periphery of the peptide segments to promote solubility in water while also driving assembly of the individual and combined components into β-sheets. The photopolymerization of PDA, as well as the sensitivity of the resulting optical properties of the polymeric material to external stimuli, highly depends on the structural organization of the assembly of amphiphilic peptide-diacetylene units into 1D-nanostructures. Solid-state NMR measurements on 13C-labeled and 15N-labeled samples show that positively charged and negatively charged peptide amphiphiles are each capable of self-assembly, but self-assembly favors antiparallel β-sheet structure. When positively and negatively charged peptide amphiphiles interact in stoichiometric solutions, cooperative coassembly dominates over self-assembly, resulting in the desired parallel β-sheet structure with a concomitant increase in structural order. These results reveal that rational placement of oppositely charged residues can control β-strand organization in a peptide amphiphile coassembly, which would have implications on the adaptive properties of stimuli-responsive biomaterials such as the peptide-PDAs studied here.

PL3 CendR peptide shows specific uptake in cultured Y79 retinoblastoma cells with nucleolar accumulation

Retinoblastoma is the most common pediatric intraocular malignant tumor affecting 1:15 000–1:20 000 live births. Even though the survival rate in developed countries is over 90 %, more efficient treatment options are needed for better vision salvage and reduction of the adverse effects. Therefore, we investigated fluorescein-labeled PL3 peptide targeting properties towards the Y79 retinoblastoma cell line in vitro. Through the application of cellular imaging and flow cytometry techniques, the PL3 peptide exhibited a rapid and specific internalization within Y79 cells, with subsequent translocation to the cell nuclei, showcasing notable accumulation in the nucleoli. This phenomenon was not present in other investigated cell lines and was not observable with similarly charged and length control peptide. However, the exact mechanism behind this Y79 cell line-specific nuclear and nucleolar targeting pattern remains elusive. In the future, this targeting process could facilitate specific treatment modalities of retinoblastoma with PL3 peptide-coupled drug delivery technologies.

What Are the Key Factors for the Detection of Peptides Using Mass Spectrometry on Boron-Doped Diamond Surfaces?

We investigated the use of boron-doped diamond (BDD) with different surface morphologies for the enhanced detection of nine different peptides by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). For the first time, we compared three different nanostructured BDD film morphologies (Continuous, Nanograss, and Nanotips) with differently terminated surfaces (-H, -O, and -F) to commercially available Ground Steel plates. All these surfaces were evaluated for their effectiveness in detecting the nine different peptides by MALDI-MS. Our results demonstrated that certain nanostructured BDD surfaces exhibited superior performance for the detection of especially hydrophobic peptides (e.g., bradykinin 1-7, substance P, and the renin substrate), with a limit of detection of down to 2.3 pM. Further investigation showed that hydrophobic peptides (e.g., bradykinin 1-7, substance P, and the renin substrate) were effectively detected on hydrogen-terminated BDD surfaces. On the other hand, the highly acidic negatively charged peptide adrenocorticotropic hormone fragment 18-39 was effectively identified on oxygen-/fluorine-terminated BDD surfaces. Furthermore, BDD surfaces reduced sodium adduct contamination significantly.

Impact of Charge State on Characterization of Large Middle-Down Sized Peptides by Tandem Mass Spectrometry.

Fragmentation trends of large peptides were characterized by five activation methods, including HCD, ETD, EThcD, 213 nm UVPD, and 193 nm UVPD. Sequence coverages and scores were assessed based on charge site, peptide sequence, and peptide size. The effect of charge state and peptide size on sequence coverage was explored for a Glu-C digest of E. coli ribosomal proteins, and linear regression analysis of the collection of peptides indicated that HCD, ETD, and EThcD have a higher dependence charge state than 193 and 213 nm UV. Four model peptides, neuromedin, glucagon, galanin, and amyloid β, were characterized in greater detail based on charge site analysis and showed a charge state dependence on sequence coverage for collision and electron-based activation methods.

The zinc absorption of the novel peptide-Zn complex in Caco-2 cells: effects of soybean peptides charge and hydrophobicity.

The supplemental effect of zinc depends not only on adequate intake, but also on how efficiently it is absorbed in the small intestine. In the present study, weak hydrophobic peptides (WHP), strong hydrophobic peptides (SHP), positively charged peptides (PCP) and negatively charged peptides (NCP) were isolated from soybean peptides (SP). The peptide-Zn complexes (PCP-Zn, NCP-Zn, WHP-Zn, SHP-Zn and SP-Zn) were prepared to compare their promotion zinc absorption capacity in the Caco-2 cells monolayers model. We found that the carboxyl, carbonyl and amino groups in peptide were the primary binding sites of Zn. Compared with zinc sulfate, the peptide-Zn complexes with different charge and hydrophobic peptides could improve zinc solubility at different pH. NCP-Zn had a lower Zn-binding capacity but a higher zinc absorption capacity compared to that of PCP-Zn in Caco-2 cells. In addition, the capacity of PCP-Zn to promote zinc absorption was lower than the control group (SP-Zn). There were no significant differences in transport rates, retention rates and uptake rates of WHP-Zn, SHP-Zn and SP-Zn. NCP-Zn could improve the activity of Zn-related enzymes, and the expression levels of PepT1 and ZnT1 were higher than other peptide-Zn complexes. The promotion zinc absorption capacity of peptide-Zn complexes was not completely dependent on the Zn-binding capacity, but also depended on the charge and hydrophobicity of peptides. © 2024 Society of Chemical Industry.