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Collagen is the most abundant protein in various connective tissues, providing mechanical integrity as well as regulating cellular activities.

Background: Collagen has been widely utilized in tissue engineering, regenerative medicine and cosmetics. Collagen of low concentrations is frequently applied to reduce the production cost, while it may result in the loss of triple helical structure and bioactivity. CD and NMR techniques have enhanced our understanding of collagen triple helix, while they require high concentrations of collagen samples. Objective: We have systematically investigated the folding and unfolding features of collagen mimetic peptides at a broad variety of concentrations in order to decipher the role of the concentration in the triple helical stability. Methods: Peptide FAM-G(POG)10 was synthesized by the solid phase synthesis method. Fluorescence spectra of peptide FAM-G(POG)10 at different concentrations were recorded. The unfolding and folding profiles of peptide FAM-G(POG)10 with concentrations varying from 1 nM to 100 μM were examined. The effect of concentration on the folding and unfolding capability of peptide FAMG( POG)10 was investigated. Results: Fluorescence characterization of peptide FAM-G(POG)10 under widely varying concentrations from 1 nM to 100 μM has revealed that concentration played a critical role in the stability of collagen peptides. The two-phase pattern of the concentration-dependent folding and unfolding curves has for the first time demonstrated the presence of a critical concentration for the collagen peptide to trigger the complete folding of the triple helix and to maintain the triple helix structure. It is noteworthy that the triple helix structure of collagen peptides was very stable at μM-level concentrations from both the folding and unfolding perspectives. Conclusion: It has significantly contributed to our understanding of collagen triple helix stability at low and ultra-low concentrations, and provided valuable and practical guidelines for the preparation of collagen-based products.

A robust MRI peptide contrast agent Gd-ICTP with superior selectivity for type I collagen has been constructed, which facilitates the non-invasive in vivo detection of hepatic fibrosis.

Protein-templated biomineralization provides a robust strategy for the synthesis of protein–rare earth fluoride hybrid nanoflowers, which display high enzymatic stability and finely tunable luminescence.

Genomic deoxyribonucleic acid (DNA) is selected as the ideal carrier for preserving and transmitting the genetic information over the course of evolution. However, the genomic DNA is constantly exposed to various endogenous and environmental threats, causing a diversity of damaged bases, lesions, mismatches and base-pair modifications in the genome, eventually leading to genomic instability and cancers. Base excision repair (BER) is the most important repair mechanism, repairing a variety of DNA damages arising from oxidation, alkylation, methylation, deamination, and hydrolysis reactions. DNA glycosylases are responsible for initiating the first step of the BER pathway through cleaving the N-glycosidic bond between the damaged base and the DNA backbone. However, abnormal DNA glycosylases are associated with a variety of diseases such as cancer, cardiovascular disease, neurological disease and inflammation, suggesting the important role of DNA glycosylases in cancer diagnosis and treatment. Therefore, it is highly desirable to monitor the activity of DNA glycosylases, gaining a deep understanding of the restoration process of damaged DNA and clinical diagnosis. Recently, a series of novel DNA glycosylases detection methods with excellent performance have been developed. In this minireview, we summarize the recent advances in DNA glycosylase assays including amplification-free assay and amplification-assisted assay. Firstly, a brief introduction of amplification-free assay for DNA glycosylase is given. Then, amplification-assisted assays for DNA glycosylases are discussed in detail. Ultimately, the conclusions and prospects of the directions of DNA glycosylase assays are provided.

The development of high-performance biocompatible batteries is pivotal in personalized health-care devices such as pacemakers and neuro-stimulators. We have for the first time developed a one-pot collagen-assisted biomineralization strategy to create hierarchical CuO nanostructures. Recombinant collagen has been demonstrated as an excellent biotemplate to delicately tune the morphologies of copper oxide mesocrystals. The initial formation of Cu(OH)2 nanoneedles gradually turned into leaf-like CuO nanostructures with branched edges and a compact middle during the biomineralization process. The as-prepared leaf-like CuO mesocrystals exhibited an attractive electrochemical performance, and may have great potential as a promising anode material for lithium-ion batteries. Notably, collagen also acts as an excellent functional agent to endow the CuO nanomaterials with high biocompatibility and bioactivity. The environmentally friendly method assisted by collagen provides novel opportunities for the construction of advanced CuO nanomaterials that have a well-controlled morphology, function and electrochemical performance, and which may have promising applications in implantable health-care electronics.

Cu2+ plays an important role as the third most abundant essential trace element in normal physiological activities and metabolism of organisms.

Water-soluble PVP-Si QDs emitting bright cyan-blue color have been easily fabricated with PVP and DAMO via the one-step hydrothermal method for TNP detection. The PVP-Si QDs show a selective fluorescence quenching toward TNP based on the combination of IFE and SQE.

γ-MnO2 nanomaterials play an essential role in the development of advanced electrochemical energy storage and conversion devices with versatile industrial applications. Herein, novel dandelion-like hollow microspheres of γ-MnO2 mesocrystals have been fabricated for the first time by a one-pot biomineralization route. Recombinant collagen with unique rod-like structure has been demonstrated as a robust template to tune the morphologies of γ-MnO2 mesocrystals, and a very low concentration of collagen can alter the nanostructures of γ-MnO2 from nanorods to microspheres. The as-prepared γ-MnO2 mesocrystals formed well-ordered hollow microspheres composed of delicate nanoneedle-like units. Among all the reported γ-MnO2 with various nanostructures, the γ-MnO2 microspheres showed the most prowess to maintain high discharge capacities after 100+ cycles. The superior electrochemical performance of γ-MnO2 likely results from its unique hierarchical micro-nano structure. Notably, the γ-MnO2 mesocrystals display high biocompatibility and cellular activity. Collagen plays a key dual role in mediating the morphology as well as endowing the biofunction of the γ-MnO2 mesocrystals. This environmentally friendly biomineralization approach using rod-like collagen as the template, provides unprecedented opportunity for the production of novel nanostructured metal oxides with superior biocompatibility and electrochemical performance, which have great potential in advanced implantable and wearable health-care electronic devices.

The construction of potent peptide probes for selectively detecting denatured collagen is crucial for a variety of widespread diseases. However, all of the denatured collagen-targeting peptide probes found till date primarily utilized the repetitive (Gly-X-Y) n sequences with exclusively imino acids Pro and Hyp in the X and Y positions, which stabilized the triple helical conformation of the peptide probes, resulting in severe obstacles for their clinical applications. A novel series of peptide probes have been constructed by incorporating nonimino acids at the X position of the (GPO)3GXO(GPO)4 sequence, while the X-site residue is varied as Tyr, Phe, Asp, and Ala, respectively. Peptide probes FAM-GYO and FAM-GFO containing aromatic residues Tyr and Phe at the X position showed similarly high binding affinity and tissue-staining efficacy as the well-established peptide probe FAM-GPO, while peptide probes FAM-GDO and FAM-GAO with the corresponding charged residue Asp and the hydrophobic residue Ala indicated much weaker binding affinity and tissue-staining capability. Furthermore, FAM-GYO and FAM-GFO could specifically detect denatured collagen in different types of mouse connective tissues and efficiently stain various human pathological tissues. We have revealed for the first time that the incorporation of nonimino acids, particularly aromatic residues at the X and Y positions of the repetitive (Gly-X-Y) n sequences, may provide a convenient strategy to create novel robust collagen-targeting peptide probes, which have promising diagnostic applications in collagen-involved diseases.

To discover drugs for toxoplasmosis with less side-effects and less probability to get drug resistance is eagerly appealed for pregnant women, infant or immunocompromised patients. In this work, using TgCDPK1 as drug target, we design a method to discover new inhibitors for CDPK1 as potential drug lead for toxoplasmosis with novel scaffolds based on the combination of 2D/3D-QSAR and scaffold-hopping methods. All the binding sites of the potential inhibitors were checked by docking method, and only the ones that docked to the most conserved sites of TgCDPK1, which make them have less probability to get drug resistance, were remained. As a result, 10 potential inhibitors within two new scaffolds were discovered for TgCDPK1 with experimentally verified inhibitory activities in micromole level. The discovery of these inhibitors may contribute to the drug development for toxoplasmosis. Besides, the pipeline which is composed in this work as the combination of QSAR and scaffold-hopping is simple, easy to repeat for researchers without need of in-depth knowledge of pharmacology to get inhibitors with novel scaffolds, which will accelerate the procedure of drug discovery and contribute to the drug repurposing study.

The development of robust collagen assays is crucial in the diagnosis and treatment of various pathological conditions. Peptide probes composed of the (Gly-Pro-Hyp)n sequences have received extensi...

γ-MnO

Type I collagen, the most abundant and arguably the most complex molecule in the human body, is an ABB heterotrimer that self-assembles to form well-defined nanofibers. We herein for the first time report the construction of peptides that could simultaneously mimic the heterotrimer composition and the self-assembly features of Type I collagen.

Aggregation-induced emission (AIE) probes have emerged as promising "turn-on" sensing tools for DNA and proteins, and the AIE biosensors conjugated with graphene oxide (GO) have shown improved selectivity. Collagen is an essential structural protein in the human body, and its degraded products are involved in a plethora of severe diseases. Collagen has a high content of charged amino acids, while EOG represents one of the most abundant charged triplets in Type I collagen. We, herein, for the first time report the construction of a GO-aided AIE biosensor for the detection of charged collagen peptides. We have shown that an AIE fluorophore TPE conjugated with a triple helical peptide TPE-PRG possesses strong fluorescence due to the restriction of intramolecular rotation of TPE in the trimer state. The adsorption of the probe TPE-PRG by GO leads to efficient fluorescence quenching, while the addition of target collagen peptide EOG releases the probe peptide from the GO surface and recovers its fluorescence. We have demonstrated that the TPE-PRG/GO complex provides a highly specific "turn-on" sensing platform for the target collagen peptide with a typical charged amino acid-rich sequence. The assay has shown little interference from other biomolecules, and it can also effectively distinguish the target charged collagen peptide from its single amino acid mutant type. The development of robust analytical assays for charged collagen peptides could pronouncedly extend our capability to investigate the pathology of collagen diseases, showing great potential for their molecular diagnosis.

Osteogenesis imperfecta (OI) is a hereditary bone disorder with various phenotypes ranging from mild multiple fractures to perinatal lethal cases, and it mainly results from the substitution of Gly by a bulkier residue in type I collagen. Triple-helical peptide models of Gly mutations have been widely utilized to decipher the etiology of OI, although these studies are mainly limited to characterizing the peptide features, such as stability and conformation in the solution state. Herein, we have constructed a new series of triple-helical peptides DD(GPO)5 ZPO(GPO)4 DD (Z=Ala, Arg, Asp, Cys, Glu, Ser, and Val) mimicking the most common types of observed OI cases. The inclusion of special terminal aspartic acids enables these collagen mimetic peptides to self-assemble to form nanomaterials upon the trigger of lanthanide ions. We have for the first time systematically evaluated the effect of different OI mutations on the aggregated state of collagen mimetic peptides. We have revealed that the identity of the Gly-substituting residue plays a determinant role in the morphology and secondary structure of the collagen peptide assemblies, showing that bulkier residues tend to result in a disruptive secondary structure and defective morphology, which lead to more severe OI phenotypes. These findings of osteogenesis imperfecta collagen mimetic peptides in the aggregation state provide novel perspectives on the molecular mechanism of osteogenesis imperfecta, and may aid the development of new therapeutic strategies.

Type I collagen, the most abundant and arguably the most complex molecule in the human body, is an ABB heterotrimer that self-assembles to form well-defined nanofibers. We herein for the first time report the construction of peptides that could simultaneously mimic the heterotrimer composition and the self-assembly features of Type I collagen.

Denatured collagen is a key biomarker for various critical diseases such as cancer. Peptide probes with the repetitive (Gly-Pro-Hyp)n sequences have recently been found to selectively target denatured collagen; however, thermal or UV pretreatment is required to drive the peptides into the monomer conformation, which poses a substantial challenge for clinical applications. We herein construct two peptide probes, FAM-GOO and FAM-GPP, consisting of the repetitive (Gly-Hyp-Hyp)8 and (Gly-Pro-Pro)8 sequences, respectively. The CD, fluorescence and colorimetric studies have consistently revealed that FAM-GOO showed strong capability of forming the triple helical structure, while FAM-GPP pronouncedly displayed the single stranded conformation at temperatures as low as 4 °C. The binding experiments have indicated that both peptide probes could recognize denatured collagen with high specificity, and FAM-GPP remarkably did not need the preheating treatment. The tissue staining results have shown that preheated FAM-GOO and unheated FAM-GPP could target denatured collagen in a wide variety of rat frozen and human FFPE tissue sections. Compared with antibodies specific for a certain type of collagen, both FAM-GOO and FAM-GPP act as broad-spectrum probes for the selective detection of denatured collagen of different types and from different species. Importantly, FAM-GPP possessed the unique capability of maintaining the monomer conformation by itself, thus avoiding the potential risks of the thermal or UV pretreatment. This novel peptide probe provides a handy and versatile biosensor for specifically targeting denatured collagen, which has attractive potential in the diagnosis and therapeutics of collagen-involved diseases.

Osteogenesis imperfecta (OI) is a hereditary connective tissue disorder occurring mainly due to missense mutations in collagen, which has complicated effects on stability and conformation of monomer collagen as well as the morphology and bioactivity of aggregated collagen. We have created a self-assembling collagen mimetic peptide system that for the first time facilitates simultaneous characterization of the effects of osteogenesis imperfecta mutations on stability, conformation, assembly and activity. The constructed collagen mimetic peptide with an integrin binding motif in the center and aspartic amino acids at two terminals maintains a classic triple helix structure, and self-assembles to form exquisite nanofibers characteristic of native collagen under the trigger of lanthanide ions. The biocompatible peptide–lanthanide assemblies exhibit good cell adhesion and spreading features, suggesting that this novel self-assembling peptide system can simultaneously well mimic the triple helix structure, and the assembly and function of native collagen. The introduction of a Gly–Ala mutation in the peptide has been found to result in a disrupted triple helix structure, abnormal assembly and loss of activity. These results have demonstrated that the self-assembling collagen peptide system provides a robust platform to evaluate OI mutations at multiple levels varying from solution structure to assembly and function. The system may be expanded to investigate a variety of collagen interactions, and it would greatly contribute to our insights into collagen-related diseases and therapies.

The enzyme OtsA (trehalose-P synthase) plays a critical role in the biosynthesis of trehalose, which is a nonreducing disaccharide that plays important functions in many organisms. By using light scattering technique, we discovered that OtsA in Arthrobacter strain A3 polymerized in the presence of divalent metal ions (Mg2+ or Ca2+), and the kinetics of the assembly was dependent on their concentrations. We identified potential compounds that can affect the kinetics of the polymerization, particularly, heparin, which acts as a very promising inhibitor of the polymerization. The OtsA assembly turns out to be a very delicate process that is finely regulated by pH. OtsA may be in the polymerized form at physiological pH in vivo, suggesting a more complicated mechanism of the enzyme. These unique properties of OtsA provide novel insights into the molecular mechanism of the biosynthesis of trehalose.

Due to their extraordinary features such as large surface areas, high porosity, and active surface functional groups, protein-inorganic nanoflowers have gained increasing attention. We have for the first time synthesized collagen-zinc phosphate hybrid nanoflowers via a mild biomineralization process. The incubation of 1.8 mM Zn(NO3)2 with 0.2 wt% of collagen protein in 20 mM PBS at 25 °C for 24 hours leads to the formation of exquisite flower-like nanomaterials with branched petals. Collagen may initiate the nucleation of the zinc phosphate crystals to form a framework of petals, and then glue the petals together. The synthesized collagen-Zn3(PO4)2 nanoflowers display catalytic activity towards water oxidation. The triple helical structure and repetitive (Gly-X-Y)n sequence pattern may endow collagen with a distinct capability to provide a superior biotemplate for producing hierarchical hybrid nanomaterials. The creation of collagen-zinc phosphate hybrid nanoflowers by our facile and green tactics significantly enhances our capability to construct hopeite-based functional materials.

The extraordinary biological and structural features of collagen has aroused great interest to construct collagen mimetic materials with promising applications in tissue engineering and regenerative medicine. We herein report the construction of a novel family of bolaamphiphile-like collagen mimetic peptides composed of only natural amino acids. The peptides possess two hydrophilic heads consisting of charged aspartic acids, which are connected by the repetitive triple helical sequences typical of collagen. We have demonstrated that the terminal aspartic acids play an essential role in the self-assembly of bolaamphiphile-like collagen mimetic peptides, and their presence is required to trigger the self-assembly. We have shown that bolaamphiphile-like collagen mimetic peptides containing either (GPO)m or (PPG)m triplets consistently result in well-ordered nanospheres, suggesting that the amino acid content of collagen peptides may not affect their appropriate self-assembly. Both peptides D2(GPO)7D2 and D2(PPG)12D2 steadily lead to exquisite nanospheres under a broad range of pH conditions, indicating that these bolaamphiphile-like collagen mimetic peptides with charged aspartic acids at both terminals may provide a facile peptide-based approach to construct well-defined nanostructures. Bolaamphiphile-like peptides would greatly expand current strategies for self-assembling collagen peptides, aiding the discovery of improved functional biomaterials.

Controlled fabrication of CaCO3 nanoparticles with an exquisite polymorph and morphology has received intensive attention due to their broad industrial and biomedical applications. Herein we have created trypsin–CaCO3 hybrid nanoparticles with a well-controlled morphology and a crystal phase via a very mild biomineralization process. The incubation of a mixture of sodium carbonate and calcium chloride at room temperature using trypsin as the biotemplate leads to predominantly calcite mesocrystals with porous sphere-like nanostructures. The trypsin–CaCO3 hybrid nanospheres behave as superior nano-biocatalysts, which display high stability against thermal and chemical denaturation as well as excellent reusability. We have demonstrated that trypsin not only acts as a novel robust biotemplate to mediate the growth of CaCO3 crystals, but also enhances their biological properties as an excellent enzyme. This indicates that biomineralization may provide a facile and environmentally amiable approach to immobilize enzymes on calcium carbonate nanoparticles. Our studies could greatly enhance the understanding of the mechanism of the protein-mediated biomineralization of CaCO3, and extend the applications of CaCO3-based composite nanostructured biomaterials.

Construction of protein–inorganic hybrid materials with hierarchical nanostructures is critical for the creation of advanced multi-functional materials. We herein for the first time report the synthesis of protein–manganese phosphate hybrid nanomaterials by environmentally amiable biomineralization approach. We have demonstrated that collagen provides an excellent biotemplate to modulate the morphology of the hybrid materials, leading to exquisite nanoflowers with branched petals. In this time-dependent biomineralization process, collagen played an essential role in the production of protein–manganese phosphate hybrid materials by inducing the nucleation of manganese phosphates to form a scaffold as well as serving as a glue to hold the petals together. The as-prepared CL–Mn3(PO4)2 nanoflowers exhibited good catalytic activity towards water oxidation. The unique (Gly–X–Y)n amino acid sequences and triple helix structure may provide extraordinary capability for collagen to create hybrid nanomaterials via collagen-templated biomineralization. The single-size and high purity may endow recombinant collagen as a powerful strategy to establish superior biotemplates. This facile and green approach to produce collagen–manganese phosphate hybrid nanoflowers greatly advances our capability to construct manganese phosphates-based functional materials.

The thermal stability of triple helical structure plays a critical role in collagen biosynthesis, function and degradation. CD technique was utilized to characterize the thermal stability of synthetic collagen mimic peptides. Fluorescence spectroscopy is widely used with easy access all around the world because of its inexpensive instrumentation, low operation cost, easy operation, and high sensitivity. Here we have developed an alternative fluorescence method to detect the thermal stability of collagen mimic peptides. We have demonstrated that fluorescence spectroscopy could measure the thermal stability of collagen mimic peptides with low concentrations under different circumstances. This highly sensitive fluorescence self-quenching assay will greatly expedite the studies of sequence-dependent properties of collagen mimic peptides, and it has great potential in the application of determining the thermal stability of triple helix systems such as collagens, collectins, adiponectin, macrophage scavenger and C1q.

Collagen with a single amino acid substitution is the main cause of a plethora of heritable disorders such as Osteogenesis Imperfecta and Ehlers-Danlos syndrome. Though significant advances have been achieved in the development of protein assays, it remains very challenging to distinguish a protein with a single amino acid mutation from the wild-type protein. A novel fluorescent self-quenching assay has been constructed to detect target collagen peptides with a single amino acid mutation using two probe peptides. The hybridization of the probe peptide and the natural target collagen peptide results in a complete heterotrimer and strong fluorescence, whereas the mixture of the probe peptide and the mutation collagen sequences leads to a partial homotrimer and pronounced fluorescence self-quenching. The extent of fluorescence quenching is dependent on the identity of the residue replacing Gly following the order of Ala < Ser < Arg, while the Gly–Ala mutation causes the mildest fluorescence loss. The probe peptide-based fluorescence self-quenching assay facilitates specific detection of the target collagen sequence with a single Gly mutation at the nM level. The simultaneous utilization of both probe peptides enables efficient discrimination between different mutation peptides. To our knowledge, our work may be the first report of a robust analytical assay that can identify collagen fragments with single amino acid mutation, which will greatly contribute to deciphering the molecular mechanism of Osteogenesis Imperfecta as well as developing novel diagnostic strategies.

The synthesis of hematite mesocrystals with a tunable hierarchical nanostructure plays a critical role in the construction of improved functional materials. We have demonstrated that two recombinant collagen proteins can be used as superior biotemplates to produce hematite mesocrystals with easily tunable hierarchical nanostructures under hydrothermal conditions. Compared with previously reported proteins, collagen is able to regulate the hierarchical structures of hematite mesocrystals with a much lower concentration, and collagen can produce a richer diversity of hierarchical nanostructures, adding two novel diamond-like and sphere-like structures in addition to the three previously reported structures (spindle-like, olive-like, and ellipsoidal-like). The distinct (Gly-X-Y)n amino acid sequence pattern and triple helix structure may provide unique capability for collagen in protein-templated biomineralization. Our studies have indicated that sequence and structural differences in proteins may lead to a plethora of novel nanostructures, which would significantly contribute to the development of improved inorganic nanomaterials.