Helical Peptides Research Articles

Impact of coverage and guest residue on polyproline II helix peptide antifouling

AbstractPolyproline II (PPII) peptide sequences are recognized as promising biomaterials because of their attractive antifouling properties. However, the mechanisms behind their antifouling behavior have not been fully characterized. In this work we show that PPII peptide coverage, controlled by adsorption time, significantly reduces the fouling of bovine serum albumin (BSA, a model foulant). In addition, guest residues introduced into the PPII sequence are shown to significantly impact BSA adsorption as well as human mesenchymal stem cell (hMSC) spreading. This research will help guide future PPII peptide designs for incorporation into novel biomaterials. Graphical abstract

Coarse-Grain Model of Ultrarigid Polymer Rods Comprising Bifunctionally Linked Peptide Bundlemers.

Computationally designed homotetrameric helical peptide bundles have been functionalized at their N-termini to achieve supramolecular polymers, wherein individual bundles ("bundlemers") are the monomeric units. Adjacent bundles are linked via two covalent cross-links. The polymers exhibit a range of conformational properties, including formation of rigid-rods with micrometer-scale persistence lengths. Herein, a coarse-grained model is used to illuminate how molecular features affect the rod-like behavior of the polymers. With increasing affinity between bundlemer ends, a sharp transition in the persistence length is observed. Doubly linked chains exhibit larger persistence lengths and more robust formation of rigid-rod structures than singly linked chains. Chain stiffness increases with decreasing temperatures. Increasing the length of the cross-linker results in more flexible chains. This model provides insights into how molecular features control the structural properties of chains comprising doubly linked rigid bundlemers.

A Supramolecular Wire Able to Self‐Assemble on Gold Surface: Controlling the Film Length to Optimize the Device Lifetime and Electron Transfer Efficiency

AbstractA chemical “lego nanoset” has been used to realize different structures on gold surfaces. Three building blocks have been designed, in order to chemically link the surface and self‐assemble in an ordered manner. Self‐assembled films are arranged on a gold surface into 3D suprastructures via consecutive deposition of different mono‐layers, taken together by thymine‐adenine hydrogen bonds. Three films, composed of one, two, and three helical peptide layers, both containing a zinc‐tetraphenylporphyrin dye as an external sheet, are built and characterized by spectro‐electrochemical and spectroscopic techniques. All films are found to generate current under illumination, and their photoresponse and stability are studied as a function of the number of peptide layers. The efficiency of the photoconversion process has been correlated to the molecular organization of the porphyrin dyes in the film and to the templating role of the bridge between the porphyrin and the gold surface.

Molecular Dynamics Investigation of the Influenza Hemagglutinin Conformational Changes in Acidic pH.

The surface protein hemagglutinin (HA) of the influenza virus plays a pivotal role in facilitating viral infection by binding to sialic acid receptors on host cells. Its conformational state is pH-sensitive, impacting its receptor-binding ability and evasion of the host immune response. In this study, we conducted extensive equilibrium microsecond-level all-atom molecular dynamics (MD) simulations of the HA protein to explore the influence of low pH on its conformational dynamics. Specifically, we investigated the impact of protonation on conserved histidine residues (H1062) located in the hinge region of HA2. Our analysis encompassed comparisons between nonprotonated (NP), partially protonated (1P, 2P), and fully protonated (3P) conditions. Our findings reveal substantial pH-dependent conformational alterations in the HA protein, affecting its receptor-binding capability and immune evasion potential. Notably, the nonprotonated form exhibits greater stability compared to protonated states. Conformational shifts in the central helices of HA2 involve outward movement, counterclockwise rotation of protonated helices, and fusion peptide release in protonated systems. Disruption of hydrogen bonds between the fusion peptide and central helices of HA2 drives this release. Moreover, HA1 separation is more likely in the fully protonated system (3P) compared to nonprotonated systems (NP), underscoring the influence of protonation. These insights shed light on influenza virus infection mechanisms and may inform the development of novel antiviral drugs targeting HA protein and pH-responsive drug delivery systems for influenza.

Structural Insights into Helix–Loop–Helix Peptides for “Ligand-Targeting” Intracellular Drug Delivery via VEGF Receptor-Mediated Endocytosis

As a new alternative to antibody-drug conjugates, we developed “ligand-targeting” peptide-drug conjugates (PDCs), in which conformationally constrained helix-loop-helix (HLH) peptide M49 targeting human vascular endothelial growth factor-A (VEGF) was used as a drug carrier. The biochemical study showed that HLH peptide M49 made a complex with VEGF in the extracellular environment, and then the M49/VEGF complex interacts with the receptor on the cell surface to induce cellular internalization via the endocytic pathway. Here, we present an X-ray crystal structure of the M49/VEGF complex at 1.5 Å resolution using a protein crystal grown in microgravity. The structure illustrated the “ligand-targeting” cellular uptake mechanism for intracellular drug delivery and the molecular basis on the peptide-VEGF binding mode with tight binding and high target specificity. In addition, mutational studies and thermodynamic analysis provided information on the driving forces of the complex formation. This work would contribute to the design of mid-size molecular-targeting peptides as well as HLH peptides, advancing the research in drug discovery and chemical biology.

Antibacterial, Antibiofilm, and Anti-inflammatory Effects of a Novel Thrombin-Derived Peptide in Sepsis Models: Insights into Underlying Mechanisms.

We developed two short helical antimicrobial peptides, HVF18-a3 and its d-enantiomer, HVF18-a3-d, derived from the thrombin C-terminal peptide HVF18. These peptides exhibit potent antimicrobial activity against various bacteria by compromising both the outer and inner membranes, with low hemolytic activity. They are stable in the presence of physiological salts and human serum, exhibiting a low potential for developing drug resistance and excellent antibiofilm activity against Gram-negative bacteria. HVF18-a3-d also neutralized lipopolysaccharide (LPS) through direct binding interactions and suppressed the production of inflammatory cytokines through the inflammatory signaling pathway mediated by Toll-like receptor 4 in RAW264.7 cells stimulated with LPS. Both pre- and post-treatment with HVF18-a3-d significantly protected mice against fatal septic shock induced by carbapenem resistant Acinetobacter baumannii. These findings suggest HVF18-a3 and HVF18-a3-d are promising candidates for developing antibiotics against Gram-negative sepsis.

Membrane Fusion Mediated by Cationic Helical Peptide L-MMBen through Phosphatidylglycerol Recruitment.

Membrane fusion is the basis for many biological processes, which holds promise in biomedical applications including the creation of engineered hybrid cells and cell membrane functionalization. Extensive research efforts, including investigations into DNA zippers and carbon nanotubes, have been dedicated to the development of membrane fusion strategies inspired by natural SNARE proteins; nevertheless, achieving a delicate balance between membrane selectivity and high fusion efficiency through precise molecular engineering remains unclear. In our recent study, we successfully designed L-MMBen, a cationic helical antimicrobial peptide that exhibits remarkable antimicrobial efficacy while demonstrating moderate cytotoxicity. In this work, we demonstrate the effective and selective induction of fusion between phosphatidylglycerol (PG)-containing membranes by L-MMBen. By combining biophysical assays at the single-vesicle level with computer simulations at the molecular level, we discovered that L-MMBen can stably adsorb onto the surface of PG-containing membranes, leading to the formation of stalk structures between vesicles and ultimately resulting in membrane fusion. Furthermore, the occurrence of fusion is attributed to the unique ability of L-MMBen to recruit PG lipids and bridge adjacent vesicles. In contrast, its nonhelical counterpart DL-MMBen was found to lack this capability despite possessing an identical positive charge. These findings present an alternative molecule for achieving selective membrane fusion and provide insights for designing helical peptides with diverse applications.

Structure‐Based Design of Bicyclic Helical Peptides That Target the Oncogene β‐Catenin

AbstractThe inhibition of intracellular protein‐protein interactions is challenging, in particular, when involved interfaces lack pronounced cavities. The transcriptional co‐activator protein and oncogene β‐catenin is a prime example of such a challenging target. Despite extensive targeting efforts, available high‐affinity binders comprise only large molecular weight inhibitors. This hampers the further development of therapeutically useful compounds. Herein, we report the design of a considerably smaller peptidomimetic scaffold derived from the α‐helical β‐catenin‐binding motif of Axin. Sequence maturation and bicyclization provided a stitched peptide with an unprecedented crosslink architecture. The binding mode and site were confirmed by a crystal structure. Further derivatization yielded a β‐catenin inhibitor with single‐digit micromolar activity in a cell‐based assay. This study sheds light on how to design helix mimetics with reduced molecular weight thereby improving their biological activity.

Antimicrobial Peptides and Their Anti-Leishmanial Efficacies on Leishmania tropica Promastigotes In vitro.

Antimicrobial resistance is a real threat to humanity. Pentavalent antimonials are reported non-effective in leishmaniasis treatment today, in countries like India. New treatment options have been assessed worldwide lately. Antimicrobial peptides (AMP) are the leading antibiotic candidates due to their large spectrum, fast efficacy, and low resistance risks. Cathelicidins are the AMP with well-documented antimicrobial activities against bacteria, fungi, and protozoa, over their positively charged membranes. Here, we aim to design cathelicidine-like helical peptides (CLHP), and compare their anti-Leishmanial efficacies in vitro, with meglumine antimoniate (MA) on Leishmania tropica. A total of five study [TN-1-5] and two control (MA and non-drug) groups were formed. Cryopreserved L. tropica isolate was thawed and cultivated in Novy-MacNeal-Nicolle medium and then in RPMI. Five different CLHPs (TN1-5) were diluted in dimethyl sulphoxide. A total of 150 uL of CLHPs and MA were added into the first wells of the test plaques, followed by serial dilutions that revealed doses within 4 and 512 ug/mL. Then, 100 uL of cultures including 1x108/mL of L. tropica promastigotes were added into each well. Viability of promastigotes was checked with XTT, while the parasite count was assessed at 24th and 48th hours. TN3 was effective at 32 ug/mL. All tested CLHPs exhibited varying degrees of anti-Leishmanial activities, except TN5, even at its highest dose. TN3 showed a particular efficacy against L. tropica in vitro. Further studies including in vivo testing of the candidate's both efficacy and toxicity are essential.

Statistical analysis of the unique characteristics of secondary structures in proteins

Protein folding is a complex process influenced by the primary sequence of amino acids. Early studies focused on understanding whether the specificity or the conservation of properties of amino acids was crucial for folding into secondary structures such as α-helices, β-sheets, turns, and coils. However, with the advent of artificial intelligence (AI) and machine learning (ML), the emphasis has shifted towards the precise nature and occurrence of specific amino acids. In our study, we analyzed a large set of proteins from diverse organisms to identify unique features of secondary structures, particularly in terms of the distribution of polar, non-polar, and charged amino acid residues. We found that α-helices tend to have a higher proportion of charged and non-polar groups compared to other secondary structures and that the presence of oppositely charged amino acid residues in helices stabilizes them, facilitating the formation of longer helices. These characteristics are distinct to α-helices. This study offers valuable insights for researchers in the field of protein design, enabling the de-novo creation of short helical peptides for a range of applications. We have also developed a web server for extensive analysis of proteins from different databases. The web server is housed at https://proseqanalyser.iitgn.ac.in/

Translation of Deoxyribonucleic Acid into Synthetic Alpha Helical Peptides for Darwinian Evolution.

DNA-encoded libraries connect the phenotypes of synthetic molecules to a DNA barcode; however, most libraries do not tap into the potential of Darwinian evolution. Herein, we report a DNA-templated synthesis (DTS) architecture to make peptides that are stabilized into α-helical conformations via head-to-tail supramolecular cyclization. Using a pilot library targeting MDM2, we show that repeated screening can amplify a binder from the lowest abundance in the library to a ranking that correlates to binding affinity. The study also highlights the need to design libraries such that the chemistry avoids biases from the heterogeneous yield in DTS.

Structural investigation, computational analysis, and theoretical cryoprotectant approach of antifreeze protein type IV mutants.

Antifreeze proteins (AFPs) have unique features to sustain life in sub-zero environments due to ice recrystallization inhibition (IRI) and thermal hysteresis (TH). AFPs are in demand as agents in cryopreservation, but some antifreeze proteins have low levels of activity. This research aims to improve the cryopreservation activity of an AFPIV. In this in silico study, the helical peptide afp1m from an Antarctic yeast AFP was modeled into a sculpin AFPIV, to replace each of its four α-helices in turn, using various computational tools. Additionally, a new linker between the first two helices of AFPIV was designed, based on a flounder AFPI, to boost the ice interaction activity of the mutants. Bioinformatics tools such as ExPASy Prot-Param, Pep-Wheel, SOPMA, GOR IV, Swiss-Model, Phyre2, MODFOLD, MolPropity, and ProQ were used to validate and analyze the structural and functional properties of the model proteins. Furthermore, to evaluate the AFP/ice interaction, molecular dynamics (MD) simulations were executed for 20, 100, and 500 ns at various temperatures using GROMACS software. The primary, secondary, and 3D modeling analysis showed the best model for a redesigned antifreeze protein (AFP1mb, with afp1m in place of the fourth AFPIV helix) with a QMEAN (Swiss-Model) Z score value of 0.36, a confidence of 99.5%, a coverage score of 22%, and a p value of 0.01. The results of the MD simulations illustrated that AFP1mb had more rigidity and better ice interactions as a potential cryoprotectant than the other models; it also displayed enhanced activity in limiting ice growth at different temperatures.

Structural insights into molecular-targeting helix–loop–helix peptide against vascular endothelial growth factor-A

Mid-sized binding peptides have recently emerged as a new therapeutic modality. A helix-loop-helix (HLH) peptide was designed as a scaffold for combinatorial peptide libraries. We screened the HLH peptide libraries against human vascular endothelial growth factor-A (VEGF) to generate a peptide, VS42-LR3, which inhibited VEGF/receptor interaction and suppressed tumor growth in a murine xenograft model of human colorectal cancer. Here, we report the first crystal structure of the HLH peptide in a complex with VEGF at high resolution using space-grown protein crystals. The X-ray structural analysis revealed that the monomeric VS42-LR3 adopted an HLH structure and bound to VEGF at the VEGF receptor-binding site. Interestingly, from the site-directed mutagenesis, thermodynamic analysis, and molecular dynamic simulations, it turned out that the loop region in the non-interacting surface to VEGF affected the structural rigidity of the whole HLH to increase the binding affinity. These findings provide valuable insights for the design of more structurally stable and higher affinity mid-sized binding peptides as well as HLH peptides, that could play a crucial role in advancing molecular-targeting therapies.

Conformational Analysis and Organocatalytic Activity of Helical Stapled Peptides Containing α-Carbocyclic α,α-Disubstituted α-Amino Acids.

Conformational freedom-restricted peptides, such as stapled peptides, play a crucial role in the advancement of functional peptide development. We synthesized stapled octapeptides using α-carbocyclic α,α-disubstituted α-amino acids, particularly 3-allyloxy-1-aminocyclopentane-1-carboxylic acid, as the crosslink motifs. The organocatalytic capabilities of the synthesized stapled peptides were assessed in an asymmetric nucleophilic epoxidation reaction because the catalytic activities are known to be proportional to α-helicity. Despite incorporating side-chain crosslinks, the enantioselectivities of the epoxidation reaction catalyzed by stapled octapeptides were found to be comparable to those obtained using unstapled peptides. Interestingly, the stapled peptides using α-carbocyclic α,α-disubstituted α-amino acids demonstrated higher reactivities and stereoselectivities (up to 99% ee) compared to stapled peptides derived from (S)-α-(4-pentenyl)alanine, a commonly used motif for stapled peptides. These differences could be attributed to the increased α-helicity of the former stapled peptide in contrast to the latter, as evidenced by the X-ray crystallographic structures of their N-tert-butoxycarbonyl derivatives.

Converting the Amyloidogenic Islet Amyloid Polypeptide into a Potent Nonaggregating Peptide Ligand by Side Chain-to-Side Chain Macrocyclization.

The islet amyloid polypeptide (IAPP), also known as amylin, is a hormone playing key physiological roles. However, its aggregation and deposition in the pancreatic islets are associated with type 2 diabetes. While this peptide adopts mainly a random coil structure in solution, its secondary conformational conversion into α-helix represents a critical step for receptor activation and contributes to amyloid formation and associated cytotoxicity. Considering the large conformational landscape and high amyloidogenicity of the peptide, as well as the complexity of the self-assembly process, it is challenging to delineate the delicate interplay between helical folding, peptide aggregation, and receptor activation. In the present study, we probed the roles of helical folding on the function-toxicity duality of IAPP by restricting its conformational ensemble through side chain-to-side chain stapling via azide-alkyne cycloaddition. Intramolecular macrocyclization (i; i + 4) constrained IAPP into α-helix and inhibited its aggregation into amyloid fibrils. These helical derivatives slowed down the self-assembly of unmodified IAPP. Site-specific macrocyclization modulated the capacity of IAPP to perturb lipid bilayers and cell plasma membrane and reduced, or even fully inhibited, the cytotoxicity associated with aggregation. Furthermore, the α-helical IAPP analogs showed moderate to high potency toward cognate G protein-coupled receptors. Overall, these results indicate that macrocyclization represents a promising strategy to protect an amyloidogenic peptide hormone from aggregation and associated toxicity, while maintaining high receptor activity.

Dual Antimicrobial and Anticancer Activity of Membrane-Active Peptide BP52.

The linear undecapeptide BP52 was previously reported to have antibacterial activity against phytopathogenic bacteria species. Due to the structural similarities to naturally occurring cationic helical antimicrobial peptides, it was speculated that this peptide could potentially target microbial pathogens and cancer cells found in mammals. Consequently, this study aims to further investigate the structural and biological properties of this peptide. Our findings indicate that BP52 exhibits strong antimicrobial and anticancer activity while displaying relatively low levels of hemolytic activity. Hence, this study suggests that BP52 could be a potential lead compound for drug discovery against infectious diseases and cancer. Besides, new insights into the relationships between the structure and the multifunctional properties of antimicrobial peptides were also explored.

Structure-Based Design of Bicyclic Helical Peptides That Target the Oncogene β-Catenin.

The inhibition of intracellular protein-protein interactions is challenging, in particular, when involved interfaces lack pronounced cavities. The transcriptional co-activator protein and oncogene β‑catenin is a prime example of such a challenging target. Despite extensive targeting efforts, available high-affinity binders comprise only large molecular weight Inhibitors. This hampers the further development of therapeutically useful compounds.Herein, we report the design of a considerably smaller peptidomimetic scaffold derived from the α-helical β‑catenin-binding motif of Axin. Sequence maturation and bicyclization provided a stitched peptide with an unprecedented crosslink architecture. The binding mode and site were confirmed by a crystal structure. Further derivatization yielded a β-catenin inhibitor with single-digit micromolar activity in a cell-based assay. This study sheds a light on how to design helix mimetics with reduced molecular weight thereby improving their biological activity.

The C-terminal self-binding helical peptide of human estrogen-related receptor γ can be druggably targeted by a novel class of rationally designed peptidic antagonists.

Orphan nuclear estrogen-related receptor γ (ERRγ) has been recognized as a potential therapeutic target for cancer, inflammation and metabolic disorder. The ERRγ contains a regulatory AF2 helical tail linked C-terminally to its ligand-binding domain (LBD), which is a self-binding peptide (SBP) and serves as molecular switch to dynamically regulate the receptor alternation between active and inactive states by binding to and unbinding from the AF2-binding site on ERRγ LBD surface, respectively. Traditional ERRγ modulators are all small-molecule chemical ligands that can be classified into agonists and inverse agonists in terms of their action mechanism; the agonists stabilize the AF2 in ABS site with an agonist conformation, while the inverse agonists lock the AF2 out of the site to largely abolish ERRγ transcriptional activity. Here, a class of ERRγ peptidic antagonists was described to compete with native AF2 for the ABS site, thus blocking the active state of AF2 binding to ERRγ LBD domain. Self-inhibitory peptide was derived from the SBP-covering AF2 region and we expected it can rebind potently to the ABS site by reducing its intrinsic disorder and entropy cost upon the rebinding. Hydrocarbon stapling was employed to do so, which employed an all-hydrocarbon bridge across the [i, i + 4]-anchor residue pair in the N-terminal, middle or C-terminal region of the self-inhibitory peptide. As might be expected, it is revealed that the stapled peptides are good binders of ERRγ LBD domain and can effectively compete with the native AF2 helical tail for ERRγ ABS site, which exhibit a basically similar binding mode with AF2 to the site and form diverse noncovalent interactions with the site, thus conferring stability and specificity to the domain-peptide complexes.

Exo-chirality of the α-helix

The structure of helical polymers is dictated by the molecular chirality of their monomer units. Particularly, macromolecular helices with monomer sequence control have the potential to generate chiral topologies. In α-helical folded peptides, the sequential repetition of amino acids generates a chiral layer defined by the amino acid side chains projected outside the amide backbone. Despite being closely related to peptides’ structural and functional properties, to the best of our knowledge, a general exo-helical symmetry model has not been yet described for the α-helix. Here, we perform the theoretical, computational, and spectroscopic elucidation of the α-helix principal exo-helical topologies. Non-canonical labeled amino acids are employed to spectroscopically characterize the different exo-helical topologies in solution, which precisely match the theorical prediction. Backbone-to-chromophore distance also shows the expected impact in the exo-helices’ geometry and spectroscopic fingerprint. Theoretical prediction and spectroscopic validation of this exo-helical topological model provides robust experimental evidence of the chiral potential on the surface of helical peptides and outlines an entirely new structural scenario for the α-helix.

Helical peptide structure improves conductivity and stability of solid electrolytes.

Ion transport is essential to energy storage, cellular signalling and desalination. Polymers have been explored for decades as solid-state electrolytes by either adding salt to polar polymers or tethering ions to the backbone to create less flammable and more robust systems. New design paradigms are needed to advance the performance of solid polymer electrolytes beyond conventional systems. Here the role of a helical secondary structure is shown to greatly enhance the conductivity of solvent-free polymer electrolytes using cationic polypeptides with a mobile anion. Longer helices lead to higher conductivity, and random coil peptides show substantially lower conductivity. The macrodipole of the helix increases with peptide length, leading to larger dielectric constants. The hydrogen bonding of the helix also imparts thermal and electrochemical stability, while allowing for facile dissolution back to monomer in acid. Peptide polymer electrolytes present a promising platform for the design of next-generation ion-transporting materials.