Template Protein Research Articles

Insertions and deletions mediated functional divergence of Rossmann fold enzymes

Nucleobase-containing coenzymes are hypothesized to be relics of an early RNA-based world that preceded the emergence of proteins. Despite the importance of coenzyme-protein synergisms, their emergence and evolution remain understudied. An excellent target to address this issue is the Rossmann fold, the most catalytically diverse and abundant protein architecture in nature. We investigated two main Rossmann lineages: the nicotinamide adenine dinucleotide phosphate (NAD(P)) and the S-adenosyl methionine (SAM)- binding superfamilies. To identify the evolutionary changes that lead to a coenzyme specificity switch on these superfamilies, we performed structural and sequence-based Hidden Markov model analysis to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed that through insertions and deletions (InDels) and a residue substitution, the ancient β1-loop-α1 coenzyme-binding structure of NAD(P) could be reshaped into the SAM-binding β1-loop-α1 structure. To experimentally prove this obsevation, we removed three amino acids from the NAD(P)-binding pocket and solved the structure of the resulting mutant, revealing the characteristic loop features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD binding and acquiring SAM binding. Our results uncovered how nature may have utilized insertions and deletions to optimize the different coenzyme-binding pockets and the distinct functionalities observed for Rossmann superfamilies. This work also proposes a general mechanism by which protein templates could have been recycled through the course of evolution to adopt different coenzymes and confer distinct chemistries.

Functional Coating from Amyloid Superwetting Films.

Tailoring the hydrophilicity of solid surfaces with a strong affinity to water has been extensively explored in the last 20 years, but studies have been limited to the single function of wettability. Here, the multifunctional properties of tailored surface films are extended from exhibiting superwettability to facilitating biological activities. It is shown that amyloid fibrils can be universally coated onto various substrates, such as fabrics (non-woven organic masks), metal meshes, polyethersulfone (PES), glass, and more, endowing the resulting surfaces with excellent performance in oil/water mixture and emulsion separation, antifouling, and antifogging. Moreover, the biocompatible crosslinked amyloid fibril coatings can serve as a platform for biocatalytic activities by immobilizing enzymes, as shown in the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) oxidation and Reactive Black 5 (RB5)degradation by laccase from Trametes versicolor. The study provides a universal approach to modifying surface morphology and chemical properties via fibrous protein templates, opening the way to unexplored bio-based applications and functionalities.

Novel Co MOF with Ionic Liquid Comprised Portable Molecularly Imprinted Polymer-Based Electrochemical Sensor for the Point-of-Care Detection of a Breast Cancer Biomarker

A cobalt metal-organic framework (Co MOF)-ionic liquid (IL) nanocomposite comprising the base matrix of the electrode was developed with electropolymerized molecularly imprinted polymer (MIP) consisting of o-phenylenediamine (oPD) to detect carcinoembryonic antigen (CEA), a biomarker of breast cancer. Firstly, Co MOF was synthesized using hydrothermal synthesis, which was used as an electrically conductive skeleton and functionalized with an IL. Then, CEA was employed as a template in the electropolymerization of oPD-imprinted films on the surface of screen-printed carbon electrode (SPCE). Finally, the template protein was removed to form a molecularly imprinted film capable of qualitatively and quantitatively signaling CEA. Under optimized conditions, the sensor for CEA exhibits a wide detection range of 1.0 × 10−4−10 ng·ml−1 (0.32 V vs Ag/AgCl) with a limit of detection (LOD) and limit of quantification (LOQ) of 0.024 pg·ml−1 and 0.082 pg·ml−1, respectively. Besides, other parameters including the selectivity, reproducibility (RSD 2.2%), and recovery rate (92.16%–103.35%) are all satisfactory. The proposed strategy provides a new route for the development of rapid breast cancer diagnostic tools.

Protein Structure Prediction with AlphaFold2, How it Works, Limitations and Solution for Less number of Homotypic and Large number of Heterotypic Contacts

Knowing the protein structure helps us to investigate diseases in human beings related to abnormal or impaired folded proteins. This research provides a solution for how to identify the misbalance of homotypic and heterotypic contacts on the sequential stage. There are two methods of protein structure prediction, template based and Ab-initio models. Template based model matches the given sequence with the original sequence. Whereas, Ab-initio calculates the weight of the given sequence and identifies whether it is balanced or not. If the sequence is not in balance, it can be labeled as on the initial stage by calculating its weight. In this research, future directions to researchers are provided as how to achieve maximum accuracy in protein structure prediction.

Self-Cascade Nanoenzyme of Cupric Oxide Nanoparticles (CuO NPs) Induced in Situ Catalysis Formation of Polyelectrolyte as Template for the Synthesis of Near-Infrared Fluorescent Silver Nanoclusters and the Application in Glutathione Detection and Bioimaging

In this work, near-infrared fluorescent silver nanoclusters (Ag NCs) were prepared based on the in situ formed poly methacrylic acid (PMAA) as the template and stabilizer, which is synthesized by methacrylic acid (MAA) and hydroxyl radical (·OH) that is generated by the cascade nanoenzyme reaction of cupric oxide nanoparticles (CuO NPs). CuO NPs possess the intrinsic glutathione-like (GPx-like) and peroxidase-like (POD-like) activities, which can catalyze glutathione (GSH) and O2 to produce hydrogen peroxide (H2O2), and then transform into ·OH. The fluorescence intensity of Ag NCs decreases with the addition of GSH, because the -SH can easily anchor on the surface, resulting in the PMAA leaving the Ag NCs, and the coeffect of GSH and PMAA results in the aggregation to form larger Ag NPs. A good linear relationship between the fluorescence quenching rate and the GSH concentration was found in the range 0.01-40 μM with the detection limit 8.0 nM. The Ag NCs can be applied in the detection of GSH in the serum, as well as bioimaging of endogenous and exogenous GSH in cells with high sensitivity. Moreover, the normal and cancer cells can be distinguished through bioimaging because of the different GSH levels. The new method for the preparation of biocompatible nanoprobe based on the nanozyme tandem catalysis and the in situ formed template can avoid the direct usage of polymers or protein templates that hinder preparation and separation, providing a reliable approach for the synthesis, biosensing, and bioimaging of nanoclusters.

Molecular modeling, molecular dynamics simulation, and essential dynamics analysis of grancalcin: An upregulated biomarker in experimental autoimmune encephalomyelitis mice

The experimental autoimmune encephalomyelitis mouse model is the most commonly used animal model, and it best represents multiple sclerosis. Grancalcin (GCA) was discovered to be upregulated in EAE mice. GCA comprises 220 amino acids that have been assigned the UniprotKB ID Q8VC88. It is a calcium-binding protein that helps neutrophils adhere to fibronectin and the formation of focal adhesions. However, the protein data bank does not contain the crystal structure of mouse GCA. The current study aims to analyze the structural and physicochemical properties of GCA. Mouse GCA showed a high percentage identity (87%) with the crystal structure of des (1–52) grancalcin with bound calcium (chain A) from Homo sapiens identified by its PDB id 1k94_A. Using the SWISS-MODEL server, we used 1k94_A as a template protein to model the mouse GCA protein. Compared to the template structure 1K94, three potential binding sites for calcium-binding have been proposed, ranging from 13 to 20, 80 to 91, and 109 to 120 amino acids. On an i5 personal computer with 8GB of RAM, GROMACS 2020.1 was utilized to run a 100 ns molecular dynamics (MD) simulation. RMSD, Rg, and RMSF analysis of an MD simulation trajectory indicate a stable and compact state throughout the simulation period of modeled proteins. We found that GCA is primarily alpha helical (Class 1), with eight alpha helices. The essential dynamics analysis captures PCA and SASA, culminating in the biological motions that correspond to the last 1000 frames. These findings will aid the development of potential inhibitors as well as the determination of binding pockets and residues for drug-like molecules.

A dual-responsive electrochemical biosensor based on artificial protein imprinted polymers and natural hyaluronic acid for sensitive recognition towards biomarker CD44

Two main challenges for biosensors facing may be analytical sensitivity and selectivity. Ultra-highly specific protein molecularly imprinted polymers (MIPs) with characteristics of easy preparation, chemical stability, convenient assembly with transducer, offer unprecedented opportunities for high-performance recognition towards targets. A dual-responsive electrochemical biosensor was demonstrated, where integrated both artificial protein MIPs and natural hyaluronic acid (HA) probes into one flexible electrode to detect biomarker CD44. Their efficacy towards target of two types of probes were elucidated and contrasted. Dual-channel screen-printed electrodes (SPEs) were designed innovatively. As one responsive channel, protein MIPs were synthesized through a polymerization procedure between functional monomer of alginate gel, a biocompatible antifouling chemical, and template protein CD44. Natural HA probes were immobilized to produce the other responsive channel. Such binary channels realized highly sensitive and selective target quantifying with lower limit of detections (LODs) of 1.41 × 10−5 ng/mL for MIPs-based and 1.51 × 10−5 ng/mL for HA-based channels, attributing to superior specificity, antifouling property and biocompatibility of MIPs and HA, satisfying stability of SPEs, high sensitivity of electrochemical techniques. The dual-responsive strategy can be extended to other batched integration of functionalized materials and active biomolecules into electrochemical and electro-catalytic biosensors towards multiple components to monitor physical signs and diagnose human diseases.

Molecularly Imprinted Nanoparticles towards MMP9 for Controlling Cardiac ECM after Myocardial Infarction: A Predictive Experimental-Computational Chemistry Investigation

The recent advances in nanotechnology are revolutionizing preventive and therapeutic approaches to treating cardiovascular diseases. Controlling the extracellular matrix metalloproteinase (MMP) activation and expression in the failing human left ventricular myocardium represents a significant therapeutic target for heart disease. In this study, we used molecularly imprinting polymers (MIPs) to restore the correct balance between MMPs and their tissue inhibitors (TIMPs), and explored the potential of this technique exhaustively through chemical synthesis, physicochemical and biological characterizations, and computational chemistry methods. By molecular dynamics simulations based on classical force fields, we simulated the early stages of the imprinting process in solution disclosing the pivotal interaction established between the monomers and the MMP9 protein template. The average interaction energies of methacrylic acid (MAA) and poly (ethylene glycol) ethyl ether methacrylate (PEG) units were in the ranges 17–22 and 30–37 kcal/mol, respectively. At low coverage, the PEG monomers seemed firmly anchored to the protein surface and were not displaced by water, while only about 20% of MAA was replaced by water. The synthesis of MIPs was successfully with a monomer conversion higher than 99% and the production of spherical particles with average diameter of 344 ± 33 nm. HPLC analysis showed a specific recognition factor of MMP9 on MIPs of about 1.3. FT-IR Chemical Imaging confirmed the mechanisms necessary to generate a “selective memory” of the MIPs towards the enzyme. HPLC results indicated that the rebound amount of both TIMP1 and MMP2 to MIPs is lower than that of the template, showing a selectivity factor of 2.1 and 2.3, respectively. Preliminary tests on the effect of MIPs on H9C2 cells revealed that this treatment has no cytotoxic effects.

Atomic Details of Biomineralization Proteins Inspiring Protein Design and Reengineering for Functional Biominerals

Biominerals are extraordinary materials that provide organisms with a variety of functions to support life. The synthesis of biominerals and organization at the macroscopic level is a consequence of the interactions of these materials with proteins. The association of biominerals and proteins is very ancient and has sparked a wealth of research across biological, medical and material sciences. Calcium carbonate, hydroxyapatite, and silica represent widespread natural biominerals. The atomic details of the interface between macromolecules and these biominerals is very intriguing from a chemical perspective, considering the association of chemical entities that are structurally different. With this review I provide an overview of the available structural studies of biomineralization proteins, explored from the Protein Data Bank (wwPDB) archive and scientific literature, and of how these studies are inspiring the design and engineering of proteins able to synthesize novel biominerals. The progression of this review from classical template proteins to silica polymerization seeks to benefit researchers involved in various interdisciplinary aspects of a biomineralization project, who need background information and a quick update on advances in the field. Lessons learned from structural studies are exemplary and will guide new projects for the imaging of new hybrid biomineral/protein superstructures at the atomic level.

A Precise BSA Protein Template Developed the C, N, S Co-Doped Fe3O4 Nanolayers as Anodes for Efficient Lithium-Ion Batteries

Protein has abundant and exquisite multidimensional structures and numerous functional groups that can bind metal ions. Taking protein as a template can effectively promote the synthesis of metal oxide nanomaterials and form multidimensional network structures, which has great prospects in preparing high-performance lithium-ion battery (LIB) anode. However, these templates only act as carbon skeletons to support electrode materials. The biodiversity of proteins in precise regulation is not fully realized, limiting the application potential of protein templates. In this work, we selected biomineralized protein BSA as the template for the electrode materials synthesis, successfully regulated the nanocrystal and microstructure, and obtained a high-quality anode material with nanonetwork and ultrathin nanolayer structure. The nanocrystals of synthesized anode materials showed good uniformity and monodispersity, achieved the heteroatom doping, such as N and S elements, and in situ carbon coating. The carbon nanolayer structure formed by BSA protein improved the capacitance and stability of electrode materials, revealing a good application value for the LIB anode. This research can give us a further understanding and expand the idea of protein templates in the preparation of high-performance energy storage nanomaterials.

Native Protein Template Assisted Synthesis of Non-Native Metal-Sulfur Clusters

Metalloenzymes are the most proficient nature catalysts that are responsible for diverse biochemical transformations introducing excellent selectivity and performing at high rates, using intricate mutual relationships between metal ions and proteins. Inspired by nature, chemists started using naturally occurring proteins as templates to harbor non-native metal catalysts for the sustainable synthesis of molecules for pharmaceutical, biotechnological and industrial purposes. Therefore, metalloenzymes are the relevant targets for the design of artificial biocatalysts. The search and development of new scaffolds capable of hosting metals with high levels of selectivity could significantly expand the scope of bio-catalysis. To meet this challenge, herein, three native scaffolds: [1Fe-4Cys] (rubredoxin), [3Fe-4S] (ferredoxin), and [S2MoS2CuS2MoS2]-ORP (orange protein) protein scaffolds are case studies describing templates for the synthesis of non-native monomeric to mixed metal–sulfur clusters, which mimic native Ni containing metalloenzymes including [Ni-Fe] Hydrogenase and [Ni-Fe] CO Dehydrogenase. The non-native metal-substituted metalloproteins are not only useful for catalysis but also as spectroscopic probes.

Fabrication of efficient protein imprinted materials based on pearl necklace-like MOFs bacterial cellulose composites

The acquisition of efficient protein isolation substances is vital for proteomic research, whereas it's still challenging nowadays. Herein, an elaborately designed protein imprinted material based on a bacterial cellulose@ZIF-67 composite carrier (BC@ZIF-67) is proposed for the first time. In particular, due to the ultrafine fiber diameter and abundant hydroxyl functional groups of the bacterial cellulose, BC@ZIF-67 presented a compact arrangement structure similar to a pearl necklace, which greatly promoted template immobilization and mass transfer resistance in protein imprinting technology. Therefore, the protein-imprinted material (BC@ZIF-67@MIPs) fabricated by surface imprinting technology and template immobilization strategy could exhibit ultrahigh adsorption capacity (1017.0 mg g−1), excellent recognition (IF = 5.98) and rapid adsorption equilibrium time (50 min). In addition, based on the experiment outcomes, our team employed BC@ZIF-67@MIPs to enrich template protein in blended protein solutions and biosamples, identifying them as underlying candidates for isolating and purifying proteins.

Biomolecular Self-Assembly of Nanorings on a Viral Protein Template.

Although there have been many advances in synthesizing nanoparticles, their assembly into deterministic and controllable patterns remains a major challenge. Biological systems operate at the nanoscale, building structural components with great chemical specificity that enable the processes of life. By adapting them to our needs, it is possible to utilize well-defined and well-controlled scaffolds to produce materials with novel properties resulting from precise ordering on the nanoscale. This approach uses spatial arrangement instead of nanoparticle size, shape, or composition to control material properties through the collective interactions between neighboring nanoparticles. Here, we demonstrate the use of tobacco mosaic virus (TMV) coat protein as a template to self-assemble plasmonic nanoparticles. Surface plasmons are resonant oscillations in the free electrons of a metal that are excited through interaction with light. These plasmonic oscillations can couple together, giving rise to more complex modes like plasmonic ring resonances that can be used to tune the response to incident light. By exploiting the self-assembling properties and chemical addressability of TMV coat protein, we can utilize site-directed mutagenesis and bioconjugation strategies to produce highly symmetrical plasmonic nanorings, as evidenced by transmission electron microscopy (TEM). Thus, we show the utility of viral proteins in designing and assembling nanostructured building blocks for advanced materials.

P1434: CORRECTION OF CONGENITAL NEUTROPENIA-RELATED GENES IN NAÏVE IPS CELLS DERIVED FROM PATIENTS USING VIRAL-FREE CRISPR/CAS9 GENE-EDITING SYSTEM

Background: Severe congenital neutropenia (SCN) is a rare haematological condition with a heterogeneous genetic background that often leads to severe impairment of the immune system. For patients who do not respond to standard treatment, the only possible therapy is allogeneic haematopoietic stem cell transplantation. SCN patients have an increased risk of the development of myelodysplastic syndrome or acute myelogenous leukaemia. Rare genetic disorders, such as SCN, require prioritising bone marrow failure diagnosis and therapy tailored to individual patients. Human induced pluripotent stem (iPS) cells are a promising prospect for future cell therapy in a wide range of diseases like congenital neutropenia for which there are currently no cures or effective therapies. Aims: The aim of this study was to develop potential treatment procedure based on the use of iPS cells generated from epithelial cells of patients with congenital neutropenia. Using mutation-specific gRNA and CRISPR-Cas9 system, we made an attempt to repair patients-derived iPS cells which may have potential for renewal of the corrected granulocyte population in the bone marrow. Methods: Patient-derived epithelial cells were reprogrammed with the EBV-derived oriP/EBNA-1 system carrying transcription factors. The transition of these cells into the naïve state was accomplished with the use of cell culture media supplemented with Leukaemia inhibitory factor, an inhibitor of Protein kinase C, small molecule activators of WNT, and inhibitors of ERK1/2 and JNK signaling pathways. CRISPR-Cas9 was used to repair mutations in patients-derived naïve iPS cells. Into the target cells, a specific sgRNA combined with eSpCas9 protein and HDR template was introduced via nucleofection with Lonza 4D Nucleofector, as a CRISPR enhancer, brefedrin A was used. To verify the efficiency of transfection and gene edition, the targeted region were analysed with Sanger sequencing (Fig. 1). Results: We have generated primed and naïve iPS cells from 7 patients with different severity of neutropenic phenotype and genetic background, 4 another samples are pending. The morphology of the iPSCs line (primed cells) displayed a typical ESC-like colony appearance comprised of tightly packed cells. Alkaline phosphatase and immunocytochemical staining confirmed that the reprogrammed cells express pluripotent stem cell markers and can differentiate into the three germ layers. We successfully repaired a mutation in ELANE gene in 4 out of 30 screened colonies as well as TCIRG1 gene in 3 out of 14 screened colonies. Additionally, for further functional studies, we generated a homozygotic ELANE p.Cys55Arg mutant, that might serve as a unique model of the genotype-phenotype gradient of elastase activity. Image:Summary/Conclusion: We have successfully generated and repaired iPS cells derived from patients with congenital neutropenia which might serve as a procedure with a great potential in the field of cell therapy tailored to individual patients.

The molybdenum storage protein forms and deposits distinct polynuclear tungsten oxygen aggregates

Some N2-fixing bacteria store Mo to maintain the formation of the vital FeMo-cofactor dependent nitrogenase under Mo depleting conditions. The Mo storage protein (MoSto), developed for this purpose, has the unique capability to compactly deposit molybdate as polyoxometalate (POM) clusters in a (αβ)3 hexameric cage; the same occurs with the physicochemically related tungstate. To explore the structural diversity of W-based POM clusters, MoSto loaded under different conditions with tungstate and two site-specifically modified MoSto variants were structurally characterized by X-ray crystallography or single-particle cryo-EM. The MoSto cage contains five major locations for POM clusters occupied among others by heptanuclear, Keggin ion and even Dawson-like species also found in bulk solvent under defined conditions. We found both lacunary derivatives of these archetypical POM clusters with missing WOx units at positions exposed to bulk solvent and expanded derivatives with additional WOx units next to protecting polypeptide segments or other POM clusters. The cryo-EM map, unexpectedly, reveals a POM cluster in the cage center anchored to the wall by a WOx linker. Interestingly, distinct POM cluster structures can originate from identical, highly occupied core fragments of three to seven WOx units that partly correspond to those found in MoSto loaded with molybdate. These core fragments are firmly bound to the complementary protein template in contrast to the more variable, less occupied residual parts of the visible POM clusters. Due to their higher stability, W-based POM clusters are, on average, larger and more diverse than their Mo-based counterparts.

Fluorescence sensor based on molecularly imprinted polymers and core–shell upconversion nanoparticles@metal–organic frameworks for detection of bovine serum albumin

A novel strategy for sensing protein was proposed through combining the high selectivity of molecular imprinting technology with the excellent upconversion fluorescence of upconversion nanoparticles (UCNPs) and high specific surface area of metal–organic frameworks (MOFs). Herein, the UCNPs acted as signal reporter and MOFs were introduced to increase the rate of mass transfer. The UCNPs@MIL-100 as support material was prepared via a step-by-step method. The imprinted material-coated UCNPs@MIL-100 (UCNPs@MIL-100@MIPs) were obtained by sol–gel technique. The results showed that as the increase of the template protein concentration, the fluorescence intensity of UCNPs@MIL-100@MIPs quenched gradually, and the imprinting factor was 2.90. The linear in the range of 1.00 to 8.00 μM, and the detection limit was 0.59 μM. Therefore, the novel optosensing material is very promising for future applications.

Preparation of polyethylenimine modified molecular imprinting adsorbent with immobilized metal oxides for highly efficient ovalbumin adsorption

In this study, ZIF-based metal oxide molecularly imprinted polymers (ZIF@PEI@TiO2-MIPs) were prepared for efficient and selective separation and enrichment of ovalbumin (OVA) in egg white samples. The effects of pH, OVA concentration, ionic strength, and elution type on the adsorption process were investigated. ZIF@PEI@TiO2-MIPs have rich imprinted sites and large specific surface area. PEI provides a large amount of amino to bind TiO2. As a new type of solid phase extraction adsorbent, ZIF@PEI@TiO2-MIPs can rapidly adsorb OVA and have a large adsorption capacity. The adsorption time is 35 min, and the maximum adsorption capacity is 417.6 mg g−1. The imprinted sites of adsorbent can distinguish the template protein (OVA) from interference proteins, indicating that ZIF@PEI@TiO2-MIPs have good selectivity and adsorption specificity, and the adsorption model conforms to Langmuir adsorption model and pseudo-second-order kinetics. The adsorbent still has good adsorption performance after six adsorption and elution cycles with 0.5% cetyltrimethylammonium bromide (CTAB) as the eluent, indicating its good stability. In conclusion, ZIF@PEI@TiO2-MIPs can specifically identify OVA in complex samples, providing a large possibility for the application of glycoprotein in activities such as biology and clinical practice.

Synthesis of carbon dots-based surface protein-imprinted nanoparticles via sandwich-structured template pre-assemble and post-imprinting modification for enhanced fluorescence detection

Molecularly imprinted polymers combined with fluorescence sensing show potential application in biological analysis and diseases diagnosis. We illustrate a versatile strategy for synthesis of surface protein-imprinted nanoparticles via sandwich-structured template pre-assemble and post-imprinting modification (PIM) for enhanced fluorescence detection. Carbon dots anchored SiO2 nanoparticles are surface modified with both disulfanylpropanoic acid and vinyl groups. By amide linkage to the carboxyl terminals, protein templates are immobilized to the nanoparticles, and subsequently allowed to bind with functional monomers via strong interactions such as metal coordination and enzyme–inhibitor association. The sandwiched protein templates are surface imprinted by free radical copolymerization with other monomers. The templates are then removed from the imprinted shells by reduction of the disulfide bonds. The derived thiol groups are used for PIM of the imprinted cavities with carboxyl groups capable of electrostatic interaction with the protein rebound. For proof of concept, human serum albumin (HSA) was firstly used as a model template for surface imprinting. It was shown that binding of a Cu2+-chelating monomer to the immobilized HSA and the subsequent PIM could elevate the imprinting factor from 1.6 to 4.1 and further to 7.6, respectively. The final imprinted nanoparticles exhibited a low limit of detection of 9.8 nM and satisfactory recoveries ranging from 98.1 to 102.0% for measuring HSA in diluted human serum samples. This strategy was further confirmed by using trypsin, a typical serine protease, as another protein template, with both the Cu2+-chelating monomer and an enzyme inhibitor monomer binding to the surface coupled template.

Anion Exchange Affinity-Based Controllable Surface Imprinting Synthesis of Ultrathin Imprinted Films for Protein Recognition.

Anion exchange affinity-based controllable surface imprinting is an effective approach to overcome low imprinting efficiency and high non-specific binding capacity. The template proteins were first immobilized on the anchored tetraalkylammonium groups of the nanoparticles via anion exchange affinity-based interactions, enabling monolayer sorption using a low template concentration. The combined use of surface-initiated photoiniferter-mediated polymerization to precisely control the imprinted film thickness, allowing the formation of homogeneous binding cavities, and the construction of effective binding sites resulted in a low non-specific binding capacity and high imprinting efficiency. The obtained imprinted films benefited from the anion exchange mechanism, exhibiting a higher imprinting factor and faster binding rate than the reference material. Binding tests revealed that the binding strength and selective recognition properties could be tuned to a certain extent by adjusting the NaCl concentration. Additionally, in contrast to the harsh template elution conditions of the covalent immobilization approach, over 80% of the template molecules were readily removed from the imprinted films using supersonic elution with an aqueous mixture of NaCl and HAc. Introducing template immobilization by anion exchange interactions to the synthesis of imprinted materials may provide a new approach for effective biomacromolecular imprinting.

Investigation on the interaction of pyrethroid pesticides to estrogen receptor alpha through computational and experimental methods

Pyrethroid insecticides are a group of widely used bio-mimetic synthetic pesticides. However, recent studies reported that they could have an accumulation effect in human which may cause series of health problems. Estrogen receptors (ER) are a class of nuclear receptors that are vital in proper physiological behavior of estrogens. To investigate the reproductive toxicity of pyrethroids, homology modeling, molecular docking, molecular dynamic simulations (MDs) were conducted to explore the interaction between pyrethroids and ERα from atomic scale. The human ERα (2YJA) was selected as a template protein for homology modeling. Then eight typical pyrethroids and positive control estradiol were docked to the modeled protein. The highest scoring bifenthrin and the lowest scoring permethrin were chosen for in-depth analysis. MDs showed that the complex formed by permethrin with ERα had a lower RMSD value and binding free energies compared to bifenthrin. Based on these results from microscopic dimension, exposure experiments were implemented to validate the primary conclusions. VTG concentrations in male zebrafish’s blood were significantly higher under permethrin exposure than bifenthrin, suggesting a stronger estrogenic activity and binding propensity. In this regard, the structural characteristics of molecules were analyzed, expecting to provide theoretical references for subsequent drug design and rational drug application.