Peptide Assembly Research Articles

Photoresponsive peptide materials: Spatiotemporal control of self-assembly and biological functions.

Peptides work as both functional molecules to modulate various biological phenomena and self-assembling artificial materials. The introduction of photoresponsive units to peptides allows the spatiotemporal remote control of their structure and function upon light irradiation. This article overviews the photoresponsive peptide design, interaction with biomolecules, and applications in self-assembling materials over the last 30 years. Peptides modified with photochromic (photoisomerizable) molecules, such as azobenzene and spiropyran, reversibly photo-controlled the binding to biomolecules and nanostructure formation through self-assembly. Photocleavable molecular units irreversibly control the functions of peptides through cleavage of the main chain and deprotection by light. Photocrosslinking between peptides or between peptides and other biomolecules enhances the structural stability of peptide assemblies and complexes. These photoresponsive peptides spatiotemporally controlled the formation and dissociation of peptide assemblies, gene expressions, protein-drug interactions, protein-protein interactions, liposome deformation and motility, cytoskeleton structure and stability, and cell functions by appropriate light irradiation. These molecular systems can be applied to photo-control biological functions, molecular robots, artificial cells, and next-generation smart drug delivery materials.

Tunable Chirality of Self‐Assembled Dipeptides Mediated by Bipyridine Derivative

AbstractSupramolecular peptide assemblies have been widely used for the development of biomedical, catalytical, and optical materials with chiral nanostructures in view of the intrinsic chirality of peptides. However, the assembly pathway and chiral transformation behavior of various peptides remain largely elusive especially for the transient assemblies under out‐of‐equilibrium conditions. Herein, the N‐fluorenylmethoxycarbonyl‐protected phenylalanine‐tyrosine dipeptide (Fmoc‐FY) was used as a peptide assembly platform, which showed that the assembly proceeds multistep evolution. The original spheres caused by liquid‐liquid phase separation (LLPS) can nucleate and elongate into the formation of right‐handed helices which were metastable and easily converted into microribbons. Interestingly, a bipyridine derivative can be introduced to effectively control the assembly pathway and induce the formation of thermodynamically stable right‐handed or left‐handed helices at different stoichiometric ratios. In addition, the chiral assembly can also be regulated by ultrasound or enzyme catalysis. This minimalistic system not only broadens the nucleation‐elongation mechanisms of protein aggregates but also promotes the controllable design and development of chiral biomaterials.

Tunable Chirality of Self-Assembled Dipeptides Mediated by Bipyridine Derivative.

Supramolecular peptide assemblies have been widely used for the development of biomedical, catalytical, and optical materials with chiral nanostructures in view of the intrinsic chirality of peptides. However, the assembly pathway and chiral transformation behavior of various peptides remain largely elusive especially for the transient assemblies under out-of-equilibrium conditions. Herein, the N-fluorenylmethoxycarbonyl-protected phenylalanine-tyrosine dipeptide (Fmoc-FY) was used as a peptide assembly platform, which showed that the assembly proceeds multistep evolution. The original spheres caused by liquid-liquid phase separation (LLPS) can nucleate and elongate into the formation of right-handed helices which were metastable and easily converted into microribbons. Interestingly, a bipyridine derivative can be introduced to effectively control the assembly pathway and induce the formation of thermodynamically stable right-handed or left-handed helices at different stoichiometric ratios. In addition, the chiral assembly can also be regulated by ultrasound or enzyme catalysis. This minimalistic system not only broadens the nucleation-elongation mechanisms of protein aggregates but also promotes the controllable design and development of chiral biomaterials.

Self-assembly of PEG-PPS polymers and LL-37 peptide nanomicelles improves the oxidative microenvironment and promotes angiogenesis to facilitate chronic wound healing.

Refractory diabetic wounds are associated with high incidence, mortality, and recurrence rates and are a devastating and rapidly growing clinical problem. However, treating these wounds is difficult owing to uncontrolled inflammatory microenvironments and defective angiogenesis in the affected areas, with no established effective treatment to the best of our knowledge. Herein, we optimized a dual functional therapeutic agent based on the assembly of LL-37 peptides and diblock copolymer poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS). The incorporation of PEG-PPS enabled responsive or controlled LL-37 peptide release in the presence of reactive oxygen species (ROS). LL-37@PEG-PPS nanomicelles not only scavenged excessive ROS to improve the microenvironment for angiogenesis but also released LL-37 peptides and protected them from degradation, thereby robustly increasing angiogenesis. Diabetic wounds treated with LL-37@PEG-PPS exhibited accelerated and high-quality wound healing in vivo. This study shows that LL-37@PEG-PPS can restore beneficial angiogenesis in the wound microenvironment by continuously providing angiogenesis-promoting signals. Thus, it may be a promising drug for improving chronic refractory wound healing.

Enzyme-Instructed Intracellular Peptide Assemblies.

Higher-order or supramolecular protein assemblies, usually regulated by enzymatic reactions, are ubiquitous and essential for cellular functions. This evolutionary fact has provided a rigorous scientific foundation, as well as an inspiring blueprint, for exploring supramolecular assemblies of man-made molecules that are responsive to biological cues as a novel class of therapeutics for biomedicine. Among the emerging man-made supramolecular structures, peptide assemblies, formed by enzyme reactions or other stimuli, have received most of the research attention and advanced most rapidly.In this Account, we will review works that apply enzyme-instructed self-assembly (EISA) to generate intracellular peptide assemblies for developing a new kind of biomedicine, especially in the field of novel cancer nanomedicines and modulating cell morphogenesis. As a versatile and cell-compatible approach, EISA can generate nondiffusive peptide assemblies locally; thus, it provides a unique approach to target subcellular organelles with exceptional cell selectivity. We have arranged this Account in the following way: after introducing the concept, simplicity, and uniqueness of EISA, we discuss the EISA-formed intracellular peptide assemblies, including artificial filaments, in the cell cytosol. Then, we describe the representative examples targeting subcellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and the nucleus, by enzyme-instructed intracellular peptide assemblies for potential cancer therapeutics. After that, we highlight the recent exploration of the transcytosis of peptide assemblies for controlling cell morphogenesis. Finally, we provide a brief outlook of enzyme-instructed intracellular peptide assemblies. This Account aims to illustrate the promise of EISA-generated intracellular peptide assemblies in understanding diseases, controlling cell behaviors, and developing new therapeutics from a class of less explored molecular entities, which are substrates of enzymes and become building blocks of self-assembly after the enzymatic reactions.

Hierarchical assembly of tryptophan zipper peptides into stress-relaxing bioactive hydrogels

Soft materials in nature are formed through reversible supramolecular assembly of biological polymers into dynamic hierarchical networks. Rational design has led to self-assembling peptides with structural similarities to natural materials. However, recreating the dynamic functional properties inherent to natural systems remains challenging. Here we report the discovery of a short peptide based on the tryptophan zipper (trpzip) motif, that shows multiscale hierarchical ordering that leads to emergent dynamic properties. Trpzip hydrogels are antimicrobial and self-healing, with tunable viscoelasticity and unique yield-stress properties that allow immediate harvest of embedded cells through a flick of the wrist. This characteristic makes Trpzip hydrogels amenable to syringe extrusion, which we demonstrate with examples of cell delivery and bioprinting. Trpzip hydrogels display innate bioactivity, allowing propagation of human intestinal organoids with apical-basal polarization. Considering these extensive attributes, we anticipate the Trpzip motif will prove a versatile building block for supramolecular assembly of soft materials for biotechnology and medicine.

Neutron reflection and scattering in characterising peptide assemblies

Self-assemblies of de novo designed short peptides at interface and in bulk solution provide potential platforms for developing applications in many medical and technological areas. However, characterising how bioinspired supramolecular nanostructures evolve with dynamic self-assembling processes and respond to different stimuli remains challenging. Neutron scattering technologies including small angle neutron scattering (SANS) and neutron reflection (NR) can be advantageous and complementary to other state-of-the-art techniques in tracing structural changes under different conditions. With more neutron sources now available, SANS and NR are becoming increasingly popular in studying self-assembling processes of diverse peptide and protein systems, but the difficulty in experimental manipulation and data analysis can deter beginners. This review will introduce the basic theory, general experimental setup and data analysis of SANS and NR, followed by provision of their applications in characterising interfacial and solution self-assemblies of representative peptides and proteins. SANS and NR are remarkably effective in determining the morphological features self-assembled short peptides, especially size and shape transitions as a result of either sequence changes or in response to environmental stimuli, demonstrating the unique capability of NR and SANS in unravelling the interactive processes. These examples highlight the potential of NR and SANS in supporting the development of novel short peptides and proteins as biopharmaceutical candidates in the fight against many diseases and infections that share common features of membrane interactive processes.

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-β and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated β-strand sheets forming a cross-β configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-β amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.

Molecular Mechanism of STIL Coiled-Coil Domain Oligomerization.

Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes and the pathogenesis of various diseases by controlling protein self-association and protein-protein interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered domain (STIL400-700) and a structured CCD (STIL CCD718-749). Disrupting STIL oligomerization via the CCD inhibits its activity in vivo. We describe a comprehensive biophysical and structural characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (KD = 8 ± 2 µM) and tetramerization (KD = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order oligomers result in increased thermal stability and cooperativity of association. We suggest that this complex oligomerization mechanism regulates the activated levels of STIL in the cell and during centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.

Sequence-dependent catalysis and assembly to form peptide/Au nanoenzyme for glucose and plasma GSH detecting in cancer patients

Metal ions play a pivotal role in regulating and determining the functions of proteins and peptides in nature. This study aims to investigate the regulatory role of metal ions in peptide assembly and explore the influence of sequence variations and metal ions on the structure and function of resulting peptide nanoarchitectures. Dipeptide sequences with distinct charged properties (positive and negative) and functional groups (-COOH, -NH2, and phenolic hydroxyl) were meticulously selected and co-assembled with various metal ions (Au3+, Ag+, and Pt4+). The findings highlight the crucial functional role of the phenolic hydroxyl group of tyrosine in metal ion reduction, while positively charged groups promote metal ion accumulation through electrostatic forces, facilitating co-assembly. The formation of ordered structures in Au@Fmoc-YK and Au@Fmoc-YR nanoarchitectures further validates the significant interaction among metal ions, tyrosine-OH, and positively charged NH2. Notably, these nanoarchitectures possess the unique attribute of being prepared under physiological conditions, specifically at 37 °C, without the need for organic solvents or chemical modifications of peptides. This approach offers a straightforward means of constructing diverse functional nanoarchitectures based on peptides and metal ions. Moreover, Au@Fmoc-YR exhibits good performance as a nanoenzyme for detecting glucose in complex bodily fluids and plasma GSH in tumor patients, showcasing its promising potential for medical applications.

Controlling 1D Nanostructures and Handedness by Polar Residue Chirality of Amphiphilic Peptides.

Peptide assemblies are promising nanomaterials, with their properties and technological applications being highly hinged on their supramolecular architectures. Here, how changing the chirality of the terminal charged residues of an amphiphilic hexapeptide sequence Ac-I4 K2 -NH2 gives rise to distinct nanostructures and supramolecular handedness is reported. Microscopic imaging and neutron scattering measurements show thin nanofibrils, thick nanofibrils, and wide nanotubes self-assembled from four stereoisomers. Spectroscopic and solid-state nuclear magnetic resonance (NMR) analyses reveal that these isomeric peptides adopt similar anti-parallel β-sheet secondary structures. Further theoretical calculations demonstrate that the chiral alterations of the two C-terminal lysine residues cause the formation of diverse single β-strand conformations, and the final self-assembled nanostructures and handedness are determined by the twisting direction and degree of single β-strands. This work not only lays a useful foundation for the fabrication of diverse peptide nanostructures by manipulating the chirality of specific residues but also provides a framework for predicting the supramolecular structures and handedness of peptide assemblies from single molecule conformations.

Machine Learning-Guided Adaptive Parametrization for Coupling Terms in a Mixed United-Atom/Coarse-Grained Model for Diphenylalanine Self-Assembly in Aqueous Ionic Liquids.

Precise regulation of the peptide self-assembly into ordered nanostructures with intriguing properties has attracted intense attention. However, predicting peptide assembly at atomic resolution is a challenge due to both the structural flexibility of peptides and the associated huge computational costs. A machine learning-guided adaptive parametrization method was proposed for developing a mixed atomic and coarse-grained (CG) model through a multiobjective optimization strategy. Our model incorporates the united-atom (UA) model for diphenylalanine (P) and the polarizable electrostatic-variable coarse-grained (VaCG) model for aqueous ionic liquid [BMIM]+[BF4]- solution. In this mixed model, the coupling van der Waals (vdW) interaction is addressed by introducing virtual sites (VS) in the UA model to interact with solvent CG beads. The coupling parameters, including the electrostatic parameter and vdW parameters, are automatically optimized through ML-guided adaptive parametrization. The performance of this model was tested by some microstructural properties, e.g., the average number of P-P intermolecular hydrogen bonds (HBs) and radius distribution functions (RDFs) between P and different fragments of IL, in comparison with all-atom (AA) simulations. The computational cost is significantly reduced using such a parametrization scheme, which could search tens of thousands of force-field parameter sets, while needing only a small fraction of them to be assessed with molecular dynamics (MD) simulations. We used such a mixed resolution model to investigate the self-assembly in IL-water mixtures with variants of IL concentration (X). The long-range-ordered fibril structure is formed in a pure water system (X = 0). With an increase of IL concentrations, the formation of an ordered self-assembly nanostructure is prohibited, instead forming branched fibril at X = 2 mol % or amorphous aggregates when X > 10 mol %, resulting from the interplay between π-stacking and HB interactions between P and IL. The qualitative agreement between the simulated structures and the observed morphologies in experiments indicates the applicability of ML-guided parametrization strategy in the study of complex systems, such as polymers, lipid bilayers, and polysaccharides.

Multicopper Clusters Enable Oxidative Phenol Macrocyclization (OxPM) of Peptides.

The biosynthesis of glycopeptide antibiotics such as vancomycin and other biologically active biaryl-bridged and diaryl ether-linked macrocyclic peptides includes key enzymatic oxidative phenol macrocyclization(s) of linear precursors. However, a simple and step-economical biomimetic version of this transformation remains underdeveloped. Here, we report highly efficient conditions for preparing biaryl-bridged and diaryl ether-linked macrocyclic peptides based on multicopper(II) clusters. The selective syntheses of ring models of vancomycin and the arylomycin cyclic core illustrate the potential of this technology to facilitate the assembly of complex antibiotic macrocyclic peptides, whose syntheses are considered highly challenging. The unprecedented ability of multicopper(II) clusters to chelate tethered diphenols and promote intramolecular over intermolecular coupling reactions demonstrates that copper clusters can catalyze redox transformations that cannot be accessed by smaller metal catalysts.

Chemo-immunotherapy by dual-enzyme responsive peptide self-assembling abolish melanoma

Herein, we designed Comp. 1 to simultaneously respond to two enzymes: alkaline phosphatase and matrix metalloproteinase 2, which is commonly found in highly malignant cancer cell lines containing B16–F10 murine melanoma cells and CT26 murine colon carcinoma cells. We used the regional differences in the expression levels of dual-markers to accurately release immune molecule IND into tumor microenvironment for the activation of anti-tumor related immune effects, while in-situ self-assembly occurs. The dual-enzyme response process can further regulate the peptide precursors’ self-assembly in the form of short rod-shaped nanofibers, enabling the delivery of the loaded chemotherapeutic drug HCPT into the cancer cells and further allowing the peptide assemblies to escape from lysosomes and return to cytoplasm in the form of tiny nanoparticles to induce apoptosis of cancer cells. This process does not occur in the single-positive breast cancer cell line MCF-7 or the normal hepatocytes cell line LO2, indicating the selectivity of the cancer cells exhibited using our strategy. In vivo studies revealed that Comp. 1 can effectively cooperate with chemotherapy to enhance the immunotherapy effect and induce immune responses associated with elevated pro-inflammatory cytokines in vivo to inhibit malignant tumors growth. Our dual-enzyme responsive chemo-immunotherapy strategy feasible in anti-tumor treatment, provides a new avenue for regulating peptide self-assembly to adapt to diverse tumor properties and may eventually be used for the development of novel multifunctional anti-tumor nanomedicines.

Emergence of Photomodulated Protometabolism by Short Peptide-Based Assemblies.

In the early Earth, rudimentary enzymes must have utilized the available light energy source to modulate protometabolic processes. Herein, we report the light-responsive C-C bond manipulation via short peptide-based assemblies bound to the photosensitive molecular cofactor (azo-based photoswitch) where the energy of the light source regulated the binding sites which subsequently modulated the retro-aldolase activity. In the presence of a continual source of high-energy photons, temporal realization of a catalytically more proficient state could be achieved under nonequilibrium conditions. Further, the hydrophobic surface of peptide assemblies facilitated the binding of an orthogonal molecular catalyst that showed augmented activity (promiscuous hydrolytic activity) upon binding. This latent activity was utilized for the in situ generation of light-sensitive cofactor that subsequently modulated the retro-aldolase activity, thus creating a reaction network.

Improving 2-Chlorotrityl Chloride (2-CTC) Resin Activation.

Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.

Amyloid Fibrils Formed by Short Prion-Inspired Peptides Are Metalloenzymes.

Enzymes typically fold into defined 3D protein structures exhibiting a high catalytic efficiency and selectivity. It has been proposed that the earliest enzymes may have arisen from the self-assembly of short peptides into supramolecular amyloid-like structures. Several artificial amyloids have been shown to display catalytic activity while offering advantages over natural enzymes in terms of modularity, flexibility, stability, and reusability. Hydrolases, especially esterases, are the most common artificial amyloid-like nanozymes with some reported to act as carbonic anhydrases (CA). Their hydrolytic activity is often dependent on the binding of metallic cofactors through a coordination triad composed of His residues in the β-strands, which mimic the arrangement found in natural metalloenzymes. Tyr residues contribute to the coordination of metal ions in the active center of metalloproteins; however, their use has been mostly neglected in the design of metal-containing amyloid-based nanozymes. We recently reported that four different polar prion-inspired heptapeptides spontaneously self-assembled into amyloid fibrils. Their sequences lack His but contain three alternate Tyr residues exposed to solvent. We combine experiments and simulations to demonstrate that the amyloid fibrils formed by these peptides can efficiently coordinate and retain different divalent metal cations, functioning as both metal scavengers and nanozymes. The metallized fibrils exhibit esterase and CA activities without the need for a histidine triad. These findings highlight the functional versatility of prion-inspired peptide assemblies and provide a new sequential context for the creation of artificial metalloenzymes. Furthermore, our data support amyloid-like structures acting as ancestral catalysts at the origin of life.

Noncovalent Peptide Assembly Enables Crystalline, Permutable, and Reactive Thiol Frameworks.

Though thiols are exceptionally versatile, their high reactivity has also hindered the synthesis and characterization of well-defined thiol-containing porous materials. Leveraging the mild conditions of the noncovalent peptide assembly, we readily synthesized and characterized a number of frameworks with thiols displayed at many unique positions and in several permutations. Importantly, nearly all assemblies were structurally determined using single-crystal X-ray diffraction to reveal their rich sequence-structure landscape and the cooperative noncovalent interactions underlying their assembly. These observations and supporting molecular dynamics calculations enabled rational engineering by the positive and negative design of noncovalent interactions. Furthermore, the thiol-containing frameworks undergo diverse single-crystal-to-single-crystal reactions, including toxic metal ion coordination (e.g., Cd2+, Pb2+, and Hg2+), selective uptake of Hg2+ ions, and redox transformations. Notably, we find a framework that supports thiol-nitrosothiol interconversion, which is applicable for biocompatible nitric oxide delivery. The modularity, ease of synthesis, functionality, and well-defined nature of these peptide-based thiol frameworks are expected to accelerate the design of complex materials with reactive active sites.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,β-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

Novel strategy to improve the bioactivity and anti-hydrolysis ability of oat peptides via zinc ion-induced assembling.

This study aims to use metal ion coordinating method to improve the bioactivity and anti-hydrolysis ability of bioactive peptides. We demonstrated that zinc (Zn) coordination (10:1 mass ratio of peptide to Zn, pH 6.8, 37°C) induced assembly of oat peptides, improved pancreatic lipase (PL) inhibitory activity by 30.4-36.8% and anti-hydrolysis ability against intestinal proteases by 26.5-38.2%; meanwhile, the peptide-Zn complex drastically reduced the PL affinity to the substrate. Detailed mechanism analysis showed that the high hydrophobicity (276 of fluorescent intensity) and dense eutectic structure of peptide-Zn complexes caused the hard hydrolysis of complexed peptides by proteases; in particular, the neutralized surface charges (∼-3.6mV) of complexes imparted the peptide-Zn complex high affinity towards PL (-22.3mV) thus robust PL inhibitory activity. These findings deepened our understanding of the interaction of peptides with metal elements and set the groundwork for the enhancement and protection of bioactive peptides.