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Super-selective multivalent ligand-receptor interactions display a signature step-like onset in binding when meeting a characteristic density of target receptors. Materials engineered for super-selective binding generally display a high number of flexible ligands to enhance the systems' avidity. In many biological processes, however, ligands are present in moderate copy numbers and arranged in spatio-temporal patterns. In this low-valency regime, the rigidity of the ligand-presenting architecture plays a critical role in the selectivity of the multivalent complex through decrease of the entropic penalty of binding. Exploiting the precision in spatial design inherent to the DNA nanotechnology, we engineered a library of rigid architectures to explore how valency, affinity, and nano-spacing control the presence of super-selectivity in multivalent binding. A micromolar monovalent affinity was required for super-selective binding to be observed within low-valency systems, and the transition point for stable interactions was measured at hexavalent ligand presentation, setting the limits of the low valency regime. Super-selective binding was observed for all hexavalent architectures, and, more strikingly, the ligand pattern determined the selectivity onset. Hereby, we demonstrate for the first time that nano-control of geometric patterns can be used to discriminate between receptor densities in a super-selective manner. Materials that were indistinguishable in their molecular composition and ligand valency bound with various efficacies on surfaces with constant receptor densities. We define this new phenomenon in super-selective binding as multivalent pattern recognition.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems., 【研究成果】スプレーで植物を改変 --簡便な非遺伝子組換え植物改変法の開発--. 京都大学プレスリリース. 2022-02-24.

An in silico study, using the GALAS algorithm available in ACD/PhysChem Suite, was performed to calculate the pK a(s) of various oximes with potential application as peptide coupling additives. Among the known oximes and predicted structures, OxymaPure is superior based on the pK a values calculated, confirming the results described in the literature and validating this algorithm for further use in that field. Among the nondescribed oximes, based on pK a calculation, ethyl 2-(hydroxyimino)-2-nitroacetate seems to be a potential candidate to be used as an additive during peptide coupling., The authors wish to thank Sanji K. Bhal (Advanced Chemistry Development, Inc, ACD/Labs) for the help with the review of the article manuscript.

The lack of an appropriate analytical approach characterizing metabolites from dietary proteins may prevent further studies that could clarify their health benefits. In this study, we attempted to establish a novel analytical assay of peptide metabolites from glycinin using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), in combination with the amine derivatization technique with coumarin (Cou). Cou (30 mmol/L) derivatization of peptides under rapid (30 min) and mild (25 °C, pH 8.5) conditions caused higher MS detection of the peptides as compared to nonderivatized peptides. In addition, an MS shift of the target by Cou derivatization (+202.0 m/z) can help to easily discriminate peptide metabolites in glycinin-administered blood, by comparing the MALDI-MS spectra of Cou-derivatized plasma with those of preadministered blood. After the oral administration of glycinin (100 mg/kg) to Sprague-Dawley rats, 15 di- to tetrapeptides were successfully characterized as glycinin-derived metabolites, demonstrating that the proposed Cou-tagged MALDI-MS is an appropriate characterization technique for peptide metabolites.

Hydrogels mimic the natural extracellular matrix in terms of their nanofibrous structure and large water content. However, the lack of a combination of properties including sufficient heterogeneity in the gel structure, intrinsic antimicrobial activity, and bioactivity limits the efficiency of hydrogels for tissue engineering applications. In this work, a hydrogel with a combination of these properties was fabricated by hybridizing silk fibroin with a low-molecular-weight peptide gelator. It was observed that silk fibroin and the peptide gelator assembled orthogonally in sequence. While the morphology of silk fibroin nanofibrils was not affected by the peptide gelator, silk fibroin promoted the formation of wider nanoribbons of the peptide gelator by modulating its nucleation and growth. Orthogonal assembly maintained the antimicrobial activity of the peptide gelator and the excellent biocompatibility of silk fibroin in the hybrid gel. The hybrid gel also demonstrated improved interactions with cells, an indicator of a higher bioactivity, possibly due to the heterogeneous double network structure.

Controlling oriented crystallization is key to producing bonelike composite materials with a well-organized structure. However, producing this type of composite material using synthetic biopolymers as scaffolds is challenging. Inspired by the molecular structure of collagen-I, a collagenlike peptide─(Pro-Hyp-Gly)10 (POG10)─was designed to produce self-assembled fibrils that resemble the structure of collagen-I fibrils. In addition, the oriented mineralization of HAP crystals is formed in the fibrils that reproduces a bonelike material similar to collagen-I fibril mineralization. Unlike collagen-I fibrils, POG10 fibrils do not contain gap spaces. The molecular simulation results indicate that in addition to space confinement, the molecular field generated by POG10 can also confine the orientation of HAP, enriching our understanding of physical confinement and shedding light on the design of synthetic biopolymer scaffolds for bonelike material fabrication.

Anti-Aβ therapy has dominated clinical trials for the prevention and treatment of Alzheimer's disease (AD). However, suppressing Aβ aggregation and disintegrating mature fibrils simultaneously remains a great challenge. In this work, we developed a new strategy using a charged tubular supramolecule (CTS) with pillar[5]arene as the backbone and modifying amino and carboxyl groups at the tubular terminals (noted as CTS-A, CTS-A/C, and CTS-C, respectively) to suppress Aβ fibrillation for the first time. According to the spectroscopic and microscopic characterizations, Aβ40 fibrillation can be efficiently suppressed by CTS-A in a very low inhibitor:peptide (I:P) molar ratio (1:10). A greatly alleviated cytotoxic effect of Aβ peptides after the inhibition or disaggregation process is further disclosed. The well-organized supramolecular structure drives multivalent interaction and gains enhanced efficiency on amyloid fibrillar modulation. These results open a new path for the design of supramolecules in the application of AD treatment.

Thioamide substitution of backbone peptide bonds can probe interactions along the main chain of proteins. Despite theoretical predictions of the enhanced hydrogen bonding propensities of thioamides, previous studies often do not consider the geometric constraints imposed by folded peptide secondary structure. This work addresses drawbacks in previous studies that ignored the geometry dependence and local dielectric properties of thioamide hydrogen bonding and identifies cases where thioamides may be either stronger or weaker hydrogen-bonding partners than amides.

Small-molecular targeting peptides possess features of biocompatibility, affinity, and specificity, which is widely applied in molecular recognition and detection. Moreover, peptides can be developed into highly ordered supramolecular assemblies with boosting binding affinities, diverse functions, and enhanced stabilities suitable for biosensors construction. In this Review, we summarize recent progress of peptide-based biosensors for precise detection, especially on tumor-related analysis, as well as further provide a brief overview of the progress in tumor immune-related detection. Also, we are looking forward to the prospective future of peptide-based biosensors.

Two dual stimuli-activated photosensitizers were developed, in which two or three glutathione (GSH)-responsive 2,4-dinitrobenzenesulfonate (DNBS)-substituted zinc(II) phthalocyanine units were connected via one or two cathepsin B-cleavable Gly-Phe-Leu-Gly peptide linker(s). These dimeric and trimeric phthalocyanines were fully quenched in the native form due to the photoinduced electron transfer to the DNBS substituents and the self-quenching of the phthalocyanine units. In the presence of GSH and cathepsin B, or upon internalization into A549 and HepG2 cancer cells, these probes were activated through the release of free phthalocyanine units. The intracellular fluorescence intensity was increased upon post-incubation with GSH ester or reduced upon pre-treatment with a cathepsin B inhibitor. Upon light irradiation, these photosensitizers became highly cytotoxic with IC50 values of 0.21-0.39 μM. The photocytotoxicity was also dependent on the intracellular GSH and cathepsin B levels. The results showed that these conjugates could serve as smart photosensitizers for targeted photodynamic therapy.

The identification and localization of isomeric peptide modifications is a critical requirement of the biopharmaceutical industry. Despite the ability of liquid chromatography-mass spectrometry to identify many of the common post translational modifications, the identification of isobaric or racemized peptides is confounded by modern mass spectrometry-based techniques. Here, we present a novel approach combining liquid chromatography with a high-resolution ion mobility mass spectrometry system to differentiate peptide and peptide fragments based upon their mobility and mass.

Here, a lipidated peptide Pal-pHK-pKV with self-assembly properties and the ability to provoke the disruption of the mitochondrial voltage-dependent anion channel-1 protein (VDAC1)-hexokinase-II (HK-II) complex is reported. The effects of the peptide pHK (N-terminal 15-amino acid fragment of HK-II that specifically binds VDAC1) are compared to those of a designed biomimetic amphiphilic pHK-pKV conjugate (pHK coupled with a cell-penetrating peptide pKV) and Pal-pHK-pKV (a lipidated conjugate modified with a hydrophobic palmitic (Pal) alkyl chain). The Pal-pHK-pKV exhibits a stronger interaction with the membrane as compared to pHK-pKV, which is demonstrated by the Langmuir-Blodgett technique and two-photon excitation microscopy. The amphiphilic peptide derivatives are cytotoxic to the A549 cells, but Pal-pHK-pKV is more cytotoxic. The inhibitory effects of the pHK derivatives on the A549 cells growth are investigated through induced apoptosis pathway, depolarized mitochondrial membrane potential, inhibited glycolysis, and activated caspase. The results of the immunofluorescence evidence the specific mitochondrial targeting by those derivatives.

Peptide drugs bradykinin, a leuprolide analogue, 2(PZ-128), and rapastinel are synthesized in 56-77% yield using heating-assisted liquid-phase peptide synthesis on a soluble polynorbornene support. These drugs of commercial utility and complex structures are obtained in 2-5.5 h with no epimerization and >95% purity using only 1.2 equivalents of amino acids and coupling reagents. The peptide yield and purity are comparable or superior to the reported methods.

Alzheimer's disease (AD) has become highly relevant in aging societies, yet the fundamental molecular basis for AD is still poorly understood. New tools to study the undergoing structural conformation changes of amyloid beta (Aβ) peptides, the pathogenic hallmark of AD, could play a crucial role in the understanding of the underlying mechanisms of misfolding and cytotoxicity of this peptide. It has been recently reported that Zn2+ interacts with Aβ and changes its aggregation pathway away from less harmful fibrillar forms to more toxic species. Here, we present a versatile platform based on a set of sub-10 nm nanogap electrodes for the manipulation and sensing of biomolecules in the physiological condition at a low copy number, where molecules are trapped via dielectrophoresis (DEP) across the nanogap, which also serves as a surface-enhanced Raman spectroscopy hotspot. In this study, we demonstrate that our electrode nanogap platform can be used to study the structural difference between Aβ40 and ZnAβ40 peptides at different aggregation stages in the physiologically relevant concentration and in solution phase. The Raman spectroscopic signatures of the DEP-captured neuropeptides prove the device to be attractive as a label-free bioanalytical tool.

Aggregated form of α-synuclein in the brain has been found to be the major component of Lewy bodies that are hallmarks of Parkinson's disease (PD), the second most devastating neurodegenerative disorder. We have carried out room-temperature all-atom molecular dynamics (MD) simulations of an ensemble of widely different α-synuclein1-95 peptide monomer conformations in aqueous solution. Attempts have been made to obtain a generic understanding of the local conformational motions of different repeat unit segments, namely R1-R7, of the peptide and the correlated properties of the solvent at the interface. The analyses revealed relatively greater rigidity of the hydrophobic R6 unit as compared to the other repeat units of the peptide. Besides, water molecules around R6 have been found to be less structured and weakly interacting with the peptide. These are important observations as the R6 unit with reduced conformational motions can act as the nucleation site for the aggregation process, while less structured weakly interacting water around it can become displaced easily, thereby facilitating the hydrophobic collapse of the peptide monomers and their association during the nucleation phase at higher concentrations. In addition, we demonstrated presence of doubly coordinated highly ordered as well as triply coordinated relatively disordered water molecules at the interface. We believe that while the ordered water molecules can favor water-mediated interactions between different peptide monomers, the randomly ordered ones on the other hand are likely to be expelled easily from the interface, thereby facilitating direct peptide-peptide interactions during the aggregation process.

The pandemic of acute respiratory disease in 2019 caused by highly pathogenic and infectious SARS-CoV-2 has seriously endangered human public safety. The 6-HB (HR1-HR2 complex) formation occurring in the process of spike protein-mediated membrane fusion could serve as a conserved and potential target for the design of fusion inhibitors. Based on the HR2 domain of 6-HB, we designed and synthesized 32 stapled peptides using an all-hydrocarbon peptide stapling strategy. Owing to the improved proteolytic stability and higher helical contents, the optimized stapled peptides termed SCH2-1-20 and SCH2-1-27 showed better inhibitory activities against pseudo and authentic SARS-CoV-2 compared to the linear counterpart. Of note, SCH2-1-20 and SCH2-1-27 were proved to interfere with S protein-mediated membrane fusion. Structural modeling indicated similar binding modes between SCH2-1-20 and the linear peptide. These optimized stapled peptides could serve as potent fusion inhibitors in treating and preventing SARS-CoV-2, and the corresponding SAR could facilitate further optimization.

By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.

The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC (EcSC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to EcSC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/EcSC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.

Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic ...

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [18F]AlF NOTA-K(ABM)-αvβ6-BP, where αvβ6-BP is a peptide with high affinity for the cell surface receptor integrin αvβ6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [18F]AlF NOTA-K(ABM)-αvβ6-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αvβ6-targeted uptake with continuously improving detection of αvβ6(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (t1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αvβ6-BP with copper-64 (t1/2 = 12.7 h). Two peptides were synthesized without (1) and with (2) the ABM and radiolabeled with copper-64 to yield [64Cu]1 and [64Cu]2, respectively. The affinity of [natCu]1 and [natCu]2 for the integrin αvβ6 was assessed by enzyme-linked immunosorbent assay. [64Cu]1 and [64Cu]2 were evaluated in vitro (cell binding and internalization) using DX3puroβ6 (αvβ6(+)), DX3puro (αvβ6(-)), and pancreatic BxPC-3 (αvβ6(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroβ6/DX3puro or BxPC-3 xenograft tumors. [64Cu]1 and [64Cu]2 were synthesized in ≥97% radiochemical purity. In vitro, [natCu]1 and [natCu]2 maintained low nanomolar affinity for integrin αvβ6 (IC50 = 28 ± 3 and 19 ± 5 nM, respectively); [64Cu]1 and [64Cu]2 showed comparable binding to αvβ6(+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αvβ6(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [64Cu]2 showed a 6-fold higher binding to human serum protein than [64Cu]1. In vivo, selective uptake in the αvβ6(+) tumors was observed with tumor visualization up to 72 h for [64Cu]2. A 3-5-fold higher αvβ6(+) tumor uptake of [64Cu]2 vs [64Cu]1 was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [64Cu]2 at 48 h. Incorporation of an iodophenyl-based ABM into the αvβ6-BP ([64Cu]2) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αvβ6(+) tumors that enabled visualization of αvβ6(+) tumors up to 72 h by PET/CT imaging.

Neopetrosiamide, a 28-residue peptide from Neopetrosia sp., contains three disulfide bonds and hinders mammalian tumor cell invasion. Proper connectivity of disulfide bonds is crucial for activity. Synthetic replacement of single disulfide bridges with methylene bridges gives active analogues. Pre-stapling of one ring enhances the correct formation of the remaining disulfides by reducing isomeric possibilities and possibly initiating the correct 3D fold. Cloning and expression of neopetrosiamide in E. coli affords access to the natural linear peptide.

The synthetic unnatural amino acids and their peptides as peptidomimetics have shown remarkable structural and functional properties. In the repertoire of synthetic peptides, pseudopeptides have emerged as attractive small peptidomimetics that are capable of forming the characteristic secondary structures in the solid/solution phase, as in natural peptides. This report describes the synthesis and structural analyses of novel pseudopeptides as ethylenediprolyl (etpro) tetra/hexapeptides, comprising a chiral diaminedicarboxylate scaffold. Their NMR and CD spectral analyses strongly support the formation of the β-turn-type structures in organic solvents (ACN/MeOH). Further, the single-crystal X-ray studies of tetrapseudopeptide confirm the formation of a unique self-assembly structure as β-strand type in the solid state through hydrogen bonding. Importantly, their diamine moiety influences the formation of Cu-complexes with Cu(II) ions. A tetrapseudopeptide monocarboxylate-Cu(II) complex forms the single crystal that is studied by the single-crystal X-ray diffractometer. The crystal structure of the tetrapseudopeptide-Cu(II) complex confirms the formation of the distorted square planar geometry structure, almost like the amyloid β(Aβ)-peptide-Cu(II) complex structural geometry. Hence, these etpro-pseudopeptides are emerging peptidomimatics that form β-turn types of structures and metal complexes mainly with Cu(II) ions. These molecules could be considered for the development of peptide-based catalysts and peptide-based therapeutic drug candidates.

Temporal persistence is as important for nanocarriers as spatial accuracy. However, because of the insufficient aggreagtion and short retention time of chemotherapy drugs in tumors, their clinical application is greatly limited. A drug delivery approach dependent on the sensitivity to an enzyme present in the microenvironment of the tumor is designed to exhibit different sizes in different sites, achieving enhanced drug permeability and retention to improve tumor nanotherapy efficacy. In this work, we report a small-molecule peptide drug delivery system containing both tumor-targeting groups and enzyme response sites. This system enables the targeted delivery of peptide nanocarriers to tumor cells and a unique response to alkaline phosphatase (ALP) in the tumor microenvironment to activate morphological transformation and drug release. The amphiphilic peptide AYR self-aggregated into a spherical nanoparticle structure after encapsulating the lipid-soluble model drug doxorubicin (DOX) and rapidly converted to nanofibers via the induction of ALP. This morphological transformation toward a high aspect ratio allowed rapid, as well as effective drug release to tumor location while enhancing specific toxicity to tumor cells. Interestingly, this "transformer"-like drug delivery strategy can enhance local drug accumulation and effectively inhibit drug efflux. In vitro along with in vivo experiments further proved that the permeability and retention of antitumor drugs in tumor cells and tissues were significantly enhanced to reduce toxic side effects, and the therapeutic effect was remarkably improved compared with that of nondeformable drug-loaded peptide nanocarriers. The developed AYR nanoparticles with the ability to undergo morphological transformation in situ can improve local drug aggregation and retention time at the tumor site. Our findings provide a new and simple method for nanocarrier morphology transformation in novel cancer treatments.

Bioinspired peptide assemblies are promising candidates for use as proton-conducting materials in electrochemical devices and other advanced technologies. Progress toward applications requires establishing foundational structure-function relationships for transport in these materials. This experimental-theoretical study sheds light on how the molecular structure and proton conduction are linked in three synthetic cyclic peptide nanotube assemblies that comprise the three canonical basic amino acids (lysine, arginine, and histidine). Experiments find an order of magnitude higher proton conductivity for lysine-containing peptide assemblies compared to histidine and arginine containing assemblies. The simulations indicate that, upon peptide assembly, the basic amino acid side chains are close enough to enable direct proton transfer. The proton transfer kinetics is determined in the simulations to be governed by the structure and flexibility of the side chains. Together, experiments and theory indicate that the proton mobility is the main determinant of proton conductivity, critical for the performance of peptide-based devices.

Antimicrobial resistance (AMR) has been increasing unrelentingly worldwide, thus negatively impacting human health. The discovery and development of novel antibiotics is an urgent unmet need of the hour. However, it has become more challenging, requiring increasingly time-consuming efforts with increased commercial risks. Hence, alternative strategies are urgently needed to potentiate the existing antibiotics. In this context, short cationic peptides or peptide-based antimicrobials that mimic the activity of naturally occurring antimicrobial peptides (AMPs) could overcome the disadvantages of AMPs having evolved as potent antibacterial agents. Besides their potent antibacterial efficacy, short peptide conjugates have also gained attention as potent adjuvants to conventional antibiotics. Such peptide antibiotic combinations have become an increasingly cost-effective therapeutic option to tackle AMR. This Review summarizes the recent progress for peptide-based small molecules as promising antimicrobials and as adjuvants for conventional antibiotics to counter multidrug resistant (MDR) pathogens.

Muraymycins are peptidyl nucleoside antibiotics that contain two Cβ-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-β-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by Cβ-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe2+- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe2+- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific Cβ hydroxylation of the Leu residue to generate (2S,3S)-β-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-Cβ-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

A sulfur-involved ligation for thioamide quasi-peptides was developed via amino acids and amino aldehydes coupling. The key to the transformation was the chelation of copper with imines for chiral activation and fixation. In this environment, linear polysulfur decreased the alkalinity of single sulfur anions to prevent racemization caused by the interaction between sulfur and sodium sulfide. Dipeptides, tripeptides, tetrapeptides, and the linkage between the drug and amino acids were successfully obtained.

Biological macromolecules often exhibit correlations in fluctuations involving distinct domains. This study decodes their functional implications in RNA-protein recognition and target-specific binding. The target search of a peptide along RNA in a viral TAR-Tat complex is closely monitored using atomistic simulations, steered molecular dynamics simulations, free energy calculations, and a machine-learning-based clustering technique. An anticorrelated domain fluctuation is identified between the tetraloop and the bulge region in the apo form of TAR RNA that sets a hierarchy in the domain-specific fluctuations at each binding event and that directs the succeeding binding footsteps. Thus, at each binding footstep, the dynamic partner selects an RNA location for binding where it senses a higher fluctuation, which is conventionally reduced upon binding. This event stimulates an alternate domain fluctuation, which then dictates sequential binding footstep/s and thus the search progresses. Our cross-correlation maps show that the fluctuations relay from one domain to another specific domain until the anticorrelation between those interdomain fluctuations sustains. Artificial attenuation of that hierarchical domain fluctuation inhibits specific RNA binding. The binding is completed with the arrival of a few long-lived water molecules that mediate slightly distant RNA-protein sites and finally stabilize the overall complex. The study underscores the functional importance of naturally designed fluctuating RNA motifs (bulge, tetraloop) and their interplay in dictating the directionality of the search in a highly dynamic environment.

Super-resolution microscopy via PAINT has been widely adopted in life sciences to interrogate the nanoscale architecture of many cellular structures. However, obtaining quantitative information in fixed cellular samples remains challenging because control of labeling stoichiometry is hampered in current approaches due to click-chemistry and additional targeting probes. To overcome these challenges, we have identified a small, PDZ-based, peptide-protein interaction pair that is genetically encodable and compatible with super-resolution imaging upon cellular fixation without additional labeling. Stoichiometric labeling control by genetic incorporation of this probe into the cellular vimentin network and mitochondria resulted in super-resolved 3D reconstructions with high specificity and spatial resolution. Further characterization reveals that this peptide-protein interaction is compatible with quantitative PAINT and that its binding kinetics remains unaltered upon fixation. Finally, by fusion of our probe to nanobodies against conventional expression markers, we show that this approach provides a versatile addition to the super-resolution toolbox.

Transient oligomeric intermediates in the peptide or protein aggregation pathway are suspected to be the key toxic species in many amyloid diseases, but deciphering their molecular nature has remained a challenge. Here we show that the strategy of "double-mutant cycles", used effectively in probing protein-folding intermediates, can reveal transient interactions during protein aggregation. It does so by comparing the changes in thermodynamic parameters between the wild type, and single and double mutants. We demonstrate the strategy by probing the possible transient salt bridge partner of lysine 28 (K28) in the oligomeric states of amyloid β-40 (Aβ40), the putative toxic species in Alzheimer's disease. In mature fibrils, the binding partner is aspartate 23. This interaction differentiates Aβ40 from the more toxic Aβ42, where K28's binding partner is the C-terminal carboxylate. We selectively acetylated K28 and amidated the C-terminus of Aβ40, creating four distinct variants. Spectroscopic measurements of the kinetics and thermodynamics of aggregation show that K28 and the C-terminus interact transiently in the early phases of the Aβ40 aggregation pathway. Hydrogen-deuterium exchange mass spectrometry (using a simple analysis method that we introduce here that takes into account the isotopic mass distribution) supports this interpretation. It is also supported by cellular toxicity measurements, suggesting possible similarities in the mechanisms of toxicity of Aβ40 oligomers (which are more toxic than Aβ40 fibrils) and Aβ42. Our results show that double-mutant cycles can be a powerful tool for probing transient interactions during protein aggregation.

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which labeling chemistry is designed. In practice, only two functional groups are present in every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative alkylation for peptide mass spectrometry and single-molecule protein sequencing that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino-acid residues for decarboxylative labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid-labeling technology in tandem to establish a new platform for fluorosequencing.

Preserving the integrity of neuronal microtubules (MTs) has emerged as a promising strategy to inhibit the progression of neurodegenerative disorders such as Alzheimer's disease. Such a goal could be achieved by peptides that mimic the functional role of Tau, an MT-associated protein that stabilizes MTs by dynamically binding to their outer surface. This work examines the binding properties and MT-stabilizing potential of a 27-amino acid Tau oligopeptide from 300 ns Gaussian-accelerated molecular dynamics simulations and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations on octameric MT models bound to two equivalent and independent Tau peptides. Bound peptides adopted extended conformations that are highly consistent with cryo-electron microscopy reports for full-length Tau bound to MTs. Anchoring points in three consecutive tubulin subunits were identified, with a relevant contribution of the Ser419-Val435 region to α-tubulin. Tau peptides strengthen the longitudinal protein-protein contacts within the MT lattice and exert a cooperative MT-stabilizing effect in MT complexes simultaneously bonded to taxol or peloruside A. Ser phosphorylation results in a larger peptide mobility, altered interaction profiles, and MT destabilization, which are in line with the loss of MT integrity resulting from the post-translational hyperphosphorylation of Tau. Our results shed light on the MT-stabilizing potential of Tau-mimetic peptides to act as novel neuroprotective agents targeting MTs.

The effect of three dietary tannins (procyanidin B3, B6, and T2) on the bioavailability of the 32-mer gliadin-derived immunogenic peptide was evaluated. An enterocyte-like Caco-2 cell line was used to mimic the epithelial transport of the 32-mer peptide, which was modeled by kinetic parameters with a mass spectrometry approach. The hydrolysis pattern on the enterocytes was analyzed, and the released peptides were quantified during the assay. The transport flux was dose-dependent. Along with procyanidin T2 and B6, procyanidin B3 promoted a significant inhibition mainly at the 100 μM peptide concentration. The hydrolysis efficiency was affected by procyanidins, while the cleavage pattern was suggested to be promoted by brush-border membranes at the apical compartment. The ability of procyanidins to molecularly bind to immunogenic peptides able to induce an adaptive response arose as a mechanism able to modulate their bioavailability, bioaccesibility, and further T CD4+ cell activation and expansion in a celiac disease (CD) model.

Many medically important viruses are enveloped viruses, which are surrounded by a structurally conserved, host-derived lipid membrane coating. Agents that target and disrupt this membrane coating could potentially function as broad-spectrum antiviral drugs. The amphipathic α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein is one such candidate and has been demonstrated to be able to selectively rupture lipid vesicles in the size range of viruses (

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry-tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.

The interest on detailed analysis of peptide-membrane interactions is of great interest in both fundamental and applied sciences as these may relate to both functional and pathogenic events. Such interactions are highly dynamic and spatially heterogeneous, making the investigation of the associated phenomena highly complex. The specific properties of membranes and peptide structural details, together with environmental conditions, may determine different events at the membrane interface, which will drive the fate of the peptide-membrane system. Here, we use an experimental approach based on the combination of spectroscopy and fluorescence microscopy methods to characterize the interactions of the multifunctional amphiphilic peptide transportan 10 with model membranes. Our approach, based on the use of suitable fluorescence reporters, exploits the advantages of phasor plot analysis of fluorescence lifetime imaging microscopy measurements to highlight the molecular details of occurring membrane alterations in terms of rigidity and hydration. Simultaneously, it allows following dynamic events in real time without sample manipulation distinguishing, with high spatial resolution, whether the peptide is adsorbed to or inserted in the membrane.

Peptides have been extensively utilized to construct nanomaterials that display targeted structure through hierarchical assembly. The self-assembly of both rationally designed peptides derived from naturally occurring domains in proteins as well as intuitively or computationally designed peptides that form β-sheets and helical secondary structures have been widely successful in constructing nanoscale morphologies with well-defined 1-d, 2-d, and 3-d architectures. In this review, we discuss these successes of peptide self-assembly, especially in the context of designing hierarchical materials. In particular, we emphasize the differences in the level of peptide design as an indicator of complexity within the targeted self-assembled materials and highlight future avenues for scientific and technological advances in this field.

Belactosins and hormaomycins are peptide natural products containing 3-(2-aminocyclopropyl)alanine and 3-(2-nitrocyclopropyl)alanine residues, respectively, with opposite stereoconfigurations of the cyclopropane ring. Herein we demonstrate that the heme oxygenase-like enzymes BelK and HrmI catalyze the N-oxygenation of l-lysine to generate 6-nitronorleucine. The nonheme iron enzymes BelL and HrmJ then cyclize the nitroalkane moiety to the nitrocyclopropane ring with the desired stereochemistry found in the corresponding natural products. We also show that both cyclopropanases remove the 4-proS-H of 6-nitronorleucine during the cyclization, establishing the inversion and retention of the configuration at C4 during the BelL and HrmJ reactions, respectively. This study reveals the unique strategy for stereocontrolled cyclopropane synthesis in nature.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

A mechanochromic polymer, polydiacetylene, changes color upon ligand binding, being a popular material in biosensing. However, whether it can also detect ligand functions in addition to binding is left understudied. In this work, we report that the polydiacetylene can be used to determine the net charges and the mode of actions (carpet model, toroidal pore model, etc.) of antimicrobial peptides and detergents via EC50 and Hill coefficients from the colorimetric response-dose curves. This opens a potential for high-throughput peptide screening by functions, which is difficult with the conventional methods.

Disulfide bonds are a class of important post-translational modifications that play important roles in modulating the structures and functions of proteins. Therefore, the mapping of disulfide linkages in peptides and proteins is indispensable for complete structure characterization and functional studies. As disulfide bonds in protonated ions do not dissociate readily under low-energy collision-induced dissociation (CID), they are usually chemically cleaved or activated prior to mass spectrometry (MS) or tandem MS (MS/MS) analysis. In this study, we report a new method that allows the mapping of disulfide linkages in peptides and proteins through meta-chloroperoxybenzoic acid (mCPBA)-based disulfide oxidation and MS/MS. Upon oxidation, the disulfide bond is converted to a thiosulfinate group, i.e., S(═O)-S, in a rapid (>60% yield in 1 min) and highly specific approach in an aqueous phase. The thiosulfinate group is then preferentially cleaved by MS/MS. For interchain disulfide linkages, this leads to a facile peptide chain separation and the identification of disulfide-linked peptides. For intrachain disulfide linkages, collisional activation of the thiosulfinate leads to disulfide cleavage and fragmentation of the peptide backbone constrained by the disulfide loop, enabling a near-complete peptide sequencing. The mCPBA oxidation-based disulfide mapping strategy can be readily integrated with bottom-up or top-down protein analysis for comprehensive protein structure elucidation, e.g., digested lysozyme and intact human insulin.

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.

Long-lived proteins (LLPs) are prone to deterioration with time, and one prominent breakdown process is the scission of peptide bonds. These cleavages can either be enzymatic or spontaneous. In this study, human lens proteins were examined and many were found to have been cleaved on the C-terminal side of Glu and Gln residues. Such cleavages could be reproduced experimentally by in vitro incubation of Glu- or Gln-containing peptides at physiological pHs. Spontaneous cleavage was dependent on pH and amino acid sequence. These model peptide studies suggested that the mechanism involves a cyclic intermediate and is therefore analogous to that characterized for cleavage of peptide bonds adjacent to Asp and Asn residues. An increased amount of some Glu/Gln cleaved peptides in the insoluble fraction of human lenses suggests that cleavage may act to destabilize proteins. Spontaneous cleavage at Glu and Gln, as well as recently described cross-linking at these residues, can therefore be added to the similar processes affecting long-lived proteins that have already been documented for Asn and Asp residues.

Delivery of functional proteins into cells may help us understand how specific protein influences cell behavior as well as treat diseases caused by protein deficiency or loss-of-function mutations. However, protein cannot enter cells by diffusion. In this work, a novel cell biology tool for delivering recombinant proteins into mammalian cells was developed. We hijacked the intracellular transport routes used by the cholera toxin and took advantage of recent development on split intein that is compatible with denatured conditions and shows an exceptional splicing activity to deliver a protein of interest into mammalian cells. Here, we used green fluorescent protein and apoptin as proofs-of-concept. The results demonstrate that the cholera toxin B subunit alone could deliver other recombinant proteins into cells through either covalent conjugation or noncovalent interaction. Our method offers more than 10-fold better delivery efficiency than the tat cell-penetrating peptide and is selective for ganglioside-rich cells. This study adds a useful tool to the receptor-mediated intracellular targeting toolkit and opens possibility for the selective delivery of therapeutic proteins into ganglioside-rich cells.

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic but non-disulfide bonded peptides known as the orbitides has been relatively ignored. What research has been conducted has focused on discovering bioactive orbitides, with less attention to structural features. A recently discovered subfamily of the orbitides known as the PawL-Derived Peptides (PLPs) are produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the family’s structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive characterization of the PLP family. Nine PLPs were produced by solid phase peptide synthesis. Although orbitides can have antimicrobial and therapeutic effects, none of these nine PLPs showed antibacterial or antifungal activity. Their structural features were studied using solution NMR spectroscopy and seven were found to possess regions of backbone order. Ordered regions consist of β-turns, with some PLPs adopting two well-defined β-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse shapes.

Cerebrovascular dysfunction is a common phenomenon in Alzheimer's patients, where fibrinogen is a major player. With the blood-brain barrier compromised, fibrinogen gains access to the brain, where its interaction with Aβ42 results in plasmin-resistant abnormal blood clots that are deposited in the cerebral blood vessels, a condition commonly encountered in Alzheimer's disease (AD) patients called cerebral amyloid angiopathy (CAA). So far, there have been no effective therapeutics available to combat AD-associated CAA. This study reports a 13-amino acid peptide (Pα-NPGRPEPGSAGTW) as a potential inhibitor of the fibrin-Aβ42 interaction along with the property to dissolve pre-existing plasmin-resistant abnormal clots. Strikingly, the identified sequence was found to be partially similar to a fragment of the fibrinogen α-chain reported to bind Aβ42, the plasmin-resistant fibrinogen fragment (PRFF). Mechanistically, Pα interacts with Aβ42 in place of fibrinogen, thus inhibiting the toxic fibrin-Aβ42 interaction. However, it does not interfere with normal fibrin polymerization.

Radical-directed dissociation (RDD) is a powerful technique for structural characterization of peptides in mass spectrometry experiments. Prior to analysis, a radical precursor must typically be appended to facilitate generation of a free radical. To explore the use of a radical precursor that can be easily attached in a single step, we modified peptides using a "click" reaction with iodophenyl isothiocyanate. Coupling with amine functional groups proceeds with high yields, producing stable iodophenylthiourea-modified peptides. Photodissociation yields were recorded at 266 and 213 nm for the 2-, 3-, and 4-iodo isomers of the modifier and found to be highest for the 4-iodo isomer in nearly all cases. Fragmentation of the modified peptides following collisional activation revealed favorable losses of the tag, and electronic structure calculations were used to evaluate a potential mechanism involving hydrogen transfer within the thiourea group. Examination of RDD data revealed that 4-iodobenzoic acid, 4-iodophenylthiourea, and 3-iodotyrosine yield similar fragmentation patterns for a given peptide, although differences in fragment abundance are noted. Iodophenyl isothiocyanate labeling in combination with RDD can be used to differentiate isomeric amino acids within peptides, which should facilitate simplified evaluation of isomers present in complex biological samples.

We report the accelerated molecular dynamics (aMD) simulation of amyloid-β (Aβ) peptides of four different lengths (16, 28, 40, and 42 residues) and their complexes when bound to Cu(II), Fe(II), or Zn(II). 600 ns equilibrated trajectory data were analyzed for each structure from three independent 200 ns aMD simulations, generating 16 aMD trajectories. We show that the presence of a metal ion leads to reduced size and decreased mobility relative to the free peptide due to the anchoring effect of the ions. The reduced mobility was shown largely to be due to the restricted movement in N-terminal residues, most notably Asp1 and His6 that are involved in the metal-ion coordination in all cases. Significant disruption of the secondary structure and patterns of salt bridge interactions arise on the coordination of metal ions. In this regard, similarities were noted between results for Zn(II) and Fe(II), whereas results for Cu(II) are more comparable to that of the free peptides. Reweighting of free energy surfaces was carried out from aMD data to identify the properties and descriptions of local minima structures.

Peptide nucleic acids (PNAs, nucleic acid analogues with a peptide backbone rather than a phosphoribosyl backbone) have emerged as promising chemical agents in antigene or antisense therapeutics, as splicing modulators or in gene editing. Their main benefits, compared to DNA or RNA agents, are their biochemical stability and the lack of negative charges throughout the backbone, leading to negligible electrostatic interaction with the strand with which they are hybridizing. As a result, hybridization of PNA strands with DNA or RNA strands leads to higher binding energies and melting temperatures. A lack of natural transporters, however, necessitates the formation of PNA-containing chimeras or the formulation of nanoparticular cell delivery methods. Here, we set out to explore the progress made in using imaging agents based on PNAs in diagnostic applications and highlight selected developments and challenges.

Zwitterionic peptides are facile low-fouling compounds for environmental applications as they are biocompatible and fully biodegradable as their degradation products are just amino acids. Here, a set of histidine (H) and glutamic acid (E), as well as lysine (K) and glutamic acid (E) based peptide sequences with zwitterionic properties were synthesized. Both oligopeptides (KE)4K and (HE)4H were synthesized in d and l configurations to test their ability to resist the nonspecific adsorption of the proteins lysozyme and fibrinogen. The coatings were additionally tested against the attachment of the marine organisms Navicula perminuta and Cobetia marina as well as the freshwater bacterium Pseudomonas fluorescens on the developed coatings. While the peptides containing lysine performed better in protein resistance assays and against freshwater bacteria, the sequences containing histidine were generally more resistant against marine organisms. The contribution of amino acid-intrinsic properties such as side chain pKa values and hydrophobicity, as well as external parameters such as pH and salinity of fresh water and seawater on the resistance of the coatings is discussed. In this way, a detailed picture emerges as to which zwitterionic sequences show advantages in future generations of biocompatible, sustainable, and nontoxic fouling release coatings.