Binding Reaction Research Articles

A percolation-type criticality threshold controls immune protein coating of surfaces.

When a material enters the body, it is immediately attacked by hundreds of proteins, organized into complex networks of binding interactions and reactions. How do such complex systems interact with a material, "deciding" whether to attack? We focus on the "complement" system of ∼40 blood proteins that bind microbes, nanoparticles, and medical devices, initiating inflammation. We show a sharp threshold for complement activation upon varying a fundamental material parameter, the surface density of potential complement attachment points. This sharp threshold manifests at scales spanning single nanoparticles to macroscale pathologies, shown here for diverse engineered and living materials. Computational models show these behaviors arise from a minimal subnetwork of complement, manifesting percolation-type critical transitions in the complement response. This criticality switch explains the "decision" of a complex signaling network to interact with a material, and elucidates the evolution and engineering of materials interacting with the body.

Targeting Human Papillomavirus 33 E2 DNA Binding Domain With Polyphenols: Unveiling Interactions Through Biophysical and In Silico Methods.

The human papillomavirus (HPV) 33 is a high-risk strain that causes lesions with potential cancerous outcomes. Its E2 protein regulates the viral protein transcription and life cycle maintenance. The DNA binding domain (DBD) of the E2 protein plays a crucial role in the viral life cycle. The DBD region of the E2 protein is particularly interesting for targeting and finding potential inhibitors to inhibit its function or dimerization. Given the limited research on HPV 33 and its proteins, the present work delved into the interaction of two natural polyphenolic compounds, resveratrol, and baicalein, with the E2 DBD of HPV 33 using biophysical and in silico studies. Fluorescence studies of the E2 DBD-polyphenol complexes showed fluorescence quenching with a binding constant of the order of 106 M-1. Circular dichroism data reveal conformational changes upon binding with the polyphenols, possibly due to distinct binding sites of the E2 DBD. Differential scanning calorimetry exhibited higher melting temperatures for the two complexes than alone DBD, suggesting the complexes' stability. ITC experiment suggested favorable binding reactions with kd values in the micromolar range. Molecular docking and dynamic simulation studies revealed that the resveratrol binds to the helical region and baicalein near the central dimeric interface of E2 DBD with a good binding affinity, forming a stable protein-ligand complex during the run of 100 ns simulation. Therefore, the current study identifies both polyphenolic compounds as promising candidates for potential antiviral drug development.

Comparative profiling of the absorbed compounds and metabolites, and pharmacokinetic studies of Danshen-Chuanxiong herb pair in rat plasma and brain using liquid chromatography-tandem mass spectrometry

Danshen-Chuanxiong (DS-CX) was a classic herb pair commonly used to treat ischemic stroke. Nevertheless, the metabolic conversion and pharmacokinetic behavior of DS-CX in vivo remains unclear. This work aimed to reveal the in vivo metabolic behavior of DS-CX through establishing metabolic profiles and performing multicomponent pharmacokinetics analysis. The mass defect filtering (MDF) strategy integrated with UHPLC-QTOF-MS was firstly developed to characterize the metabolites of DS-CX in rats’ plasma and brain. Moreover, a sensitive UHPLC-QQQ-MS method was utilized to perform the comparative pharmacokinetic studies of major active ingredients of DS-CX in rats’ plasma. A total of 111 exogenous compounds (29 prototype compounds and 82 metabolites) were identified in rat biological samples. The major metabolic pathways were hydroxylation, methylation, deoxidation, dehydration, hydrogenation, demethylation, hydrolysis, decarboxylation and glucuronidation binding reactions. According to the results of metabolites profiling, sixteen active compounds (8 phenolic acids, 5 phthalides and 3 tanshinones) were selected as markers for further comparative pharmacokinetics study. Compared with the oral administration of DS or CX alone, the higher Cmax of salvianolic acid B, crytotanshinone and tanshinone IIA; the shorter Tmax of lithospermic acid, rosmarinic acid and tanshinone IIA; as well as the higher AUC0−∞ of ferulic acid, rosmarinic acid, salvianolic acid B, senkyunolide I and crytotanshinone, could be found after co-administration of DS-CX (P < 0.05). This study provided the overall knowledge of metabolites profiling of DS-CX in vivo, which would help to understand the effective material basis and promote the clinical application of DC-CX herb pair.

Highly Stable Biotemplated InP/ZnSe/ZnS Quantum Dots for In Situ Bacterial Monitoring.

Despite their unique optical and electrical characteristics, traditional semiconductor quantum dots (QDs) made of heavy metals or carbon are not ideally suited for biomedical applications. Cytotoxicity and environmental concerns are key limiting factors affecting the adoption of QDs from laboratory research to real-world medical applications. Recently, advanced InP/ZnSe/ZnS QDs have emerged as alternatives to traditional QDs due to their low toxicity and optical properties; however, bioconjugation has remained a challenge due to surface chemistry limitations that can lead to instability in aqueous environments. Here, we report water-soluble, biotemplated InP/ZnSe/ZnS-aptamer quantum dots (QDAPTs) with long-term stability and high selectivity for targeting bacterial membrane proteins. QDAPTs show fast binding reaction kinetics (less than 5 min), high brightness, and high levels of stability (3 months) after biotemplating in aqueous solvents. We use these materials to demonstrate the detection of bacterial membrane proteins on common surfaces using a hand-held imaging device, which attests to the potential of this system for biomedical applications.

Cofactor-dependence alteration of 7β-hydroxysteroid dehydrogenase: Enhancing one-pot synthesis efficiency of chenodeoxycholic acid to ursodeoxycholic acid through cofactor self-recycling

NAD+-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) are involved in the biosynthesis of chenodeoxycholic acid (CDCA) to ursodeoxycholic acid (UDCA). To realize the one-pot synthesis of CDCA to UDCA through NAD+-NADH cycling, we aimed to improve the binding ability of Hyphomicrobium sp. 7β-HSDH to NADH. The 7β-HSDH structure was modeled and some potential residues to improve NADH affinity near conserved cofactor binding regions were screened, including Ala22, Gln23, Asn24, Asp44, Leu45, and Asn46. The dominant mutant A22T/Q23E/L45A/N46E significantly enhanced the binding affinity for NADH, resulting in a 44.9-fold increase in its kcat/Km value. It increased enzymatic activity by 65.2-fold and catalyzed the synthesis of UDCA at a yield of 77.6 % with 5 g/L 7K-LCA and 12.5 mM NADH. Molecular dynamics simulations indicated increased interactions of mutated 7β-HSDH and the ligand NADH by their spatially reduced binding distance and reaction energy. The modified cofactor-dependence of 7β-HSDH realized efficient one-pot synthesis of CDCA to UDCA through strengthening cofactor-recycling and reducing the use of cofactor, achieving 90.1 % UDCA yield and 54.1 g/L/d spatiotemporal yield when coupled with 7α-HSDH with only 0.5 mM NAD+ as coenzyme. This work also supplies a universal cofactor-dependence engineering technique for homologous HSDH enzymes.

Binding characteristics of the major kratom alkaloid, mitragynine, towards serum albumin: spectroscopic, calorimetric, microscopic, and computational investigations

Mitragynine (MTG) is a prominent indole alkaloid that is present abundantly in Mitragyna speciosa, commonly referred to as kratom. MTG has garnered significant attention due to its selective agonistic characteristics towards opioid receptors and related analgesic effects. In the circulatory system, the in vivo efficacy of MTG is dictated by its interaction with plasma proteins, primarily human serum albumin (HSA). In the present study, we utilized a broad methodology that included spectroscopic, calorimetric, microscopic, and in silico approaches to characterize the interaction between MTG and HSA. Alterations in the UV absorption spectrum of HSA by the presence of MTG demonstrated a ground-state complexation between the protein and the ligand. The Ka values obtained for the MTG–HSA interaction were in the range 103–104 M–1 based on analysis of fluorescence and ITC data, respectively, indicating an intermediate binding affinity. The binding reaction was thermodynamically favorable as revealed by ΔH, ΔS, and ΔG values of −16.42 kJ mol−1, 39.97 J mol−1 K−1, and −28.34 kJ mol−1, respectively. Furthermore, CD spectroscopy results suggested MTG binding induced minimal effects on the structural integrity of HSA, supported by computational methods. Changes in the dimensions of HSA particles due to aggregation, as observed using atomic force microscopy in the presence of MTG. Competitive drug displacement results seemingly suggested site III of HSA located at subdomain IB as the preferred binding site of MTG, but were in inconclusive. However, docking results showed the clear preference of MTG to bind to site III, facilitated by hydrophobic (alkyl and pi-alkyl) and van der Waals forces, together with carbon hydrogen bonds. Additionally, the MTG–HSA complexation was demonstrated to be stable based on molecular dynamics analysis. The outcomes of this study shed light on the therapeutic potential of MTG and can help in the design of more effective derivatives of the compound.

Optimal design of PdAu/In2O3 catalysts for CO2 hydrogenation

Efficient catalyst design has garnered significant interest in recent decades due to its potential to address both the challenges of the greenhouse effect and energy shortages by facilitating the conversion of CO2 into valuable chemicals through catalytic reactions. To investigate maximizing the synergistic effects of supported PdAu catalysts, we conducted first-principles calculations on the activation and decomposition of CO2 and H2 on the PdAu/In2O3(110) system. The results demonstrate that the incorporation of a secondary metal (Au) into the supported Pd catalyst, in conjunction with precise control over Au concentration, exerts influence on both reactant binding energy and activation. The adsorption and activation of CO2 at the interface sites of Au4/In2O3(110) and PdAu3/In2O3(110) are not observed. The transition state for the dissociation of CO2 into *CO and *O is determined based on adsorbed CO2, providing insights into the properties of activated CO2. The Bronsted–Evans–Polanyi relation, which correlates activation barriers (Ea) with reaction energies (Er), was established for the CO2 dissociation mechanism on PdAu/In2O3(110) catalysts using equation E = 0.4Ea + 0.63. It was carried out to investigate the H2-dissociated adsorption processes and mobility energy on various PdAu/In2O3(110) catalysts. Finally, a highly efficient Pd2Au2/In2O3 catalyst for the hydrogenation of CO2 into methanol has been proposed. This research provides valuable insights into the hydrogenation of CO2 to methanol using bimetal-oxide catalysts and contributes to the optimization of the design of PdAu/In2O3 catalysts for CO2 reactions.

Additive manufacturing of alumina refractories by binder jetting

In this work, refractory components based on alumina were produced by binder jetting using a large-scale 3D printer. The formulation contained several particle fractions up to a grain size of 3 mm, equal to the printer resolution. The binder system contained fine dead burnt magnesia, milled citric acid and reactive alumina, which were added to the aggregate mixture to create the powder bed. Deionized water was deposited from the printer's nozzles and triggered the binding reaction between the magnesia and citric acid. After 24 h, the printed samples were removed from the powder bed, dried and sintered at 1600 °C for 5 h. Reactive alumina contributed to the in situ creation of magnesium aluminate spinel at high temperature. The samples were characterized in terms of Young's modulus of elasticity, bending and compressive strength in 2 directions (parallel and perpendicular to the printing direction). The broken parts were used to investigate physical properties such as the open porosity and bulk density. The microstructure was studied by means of computed tomography. Finally, powder samples were used to determine the phase composition at different stages of production by means of XRD.

Assessment of machine learning models trained by molecular dynamics simulations results for inferring ethanol adsorption on an aluminium surface

Molecular dynamics (MD) simulations can reduce our need for experimental tests and provide detailed insight into the chemical reactions and binding kinetics. There are two challenges while dealing with MD simulations: one is the time and length scale limitations, and the latter is efficiently processing the massive amount of data resulting from the MD simulations and generating the proper reaction rates. In this work, we evaluated the use of regression machine learning (ML) methods to solve these two challenges by developing a framework for ethanol adsorption on an Aluminium (Al) slab. This framework comprises three main stages: first, an all-atom molecular dynamics model; second, ML regression models; and third, validation and testing. In stage one, the adsorption of ethanol molecules on the Al surface for various temperatures, velocities and concentrations is simulated using the large-scale atomic/molecular massively parallel simulator (LAMMPS) and ReaxFF. The outcome of stage one is utilised for training, testing, and validating the predictive models in stages two and three. We developed and evaluated 28 different ML models for predicting the number of adsorbed molecules over time, including linear regression, support vector machine (SVM), decision trees, ensemble, Gaussian process regression (GPR), neural network (NN) and Bayesian hyper-parameter optimisation models. Based on the results, the Bayesian-based GPR showed the highest accuracy and the lowest training time. The developed model can predict the number of adsorbed molecules for new cases within seconds, while MD simulations take a few weeks. This adsorption rate can then be used in macroscale simulations to tackle the time and length scale limitations. The proposed numerical framework has the potential to be generalised and, therefore, contribute to future low-cost binding reaction estimations, providing a valuable tool for industry and experimentalists.

Involvement of PG1037 in the repair of 8-oxo-7,8-dihydroguanine caused by oxidative stress in Porphyromonas gingivalis.

The PG1037 gene is part of the uvrA-PG1037-pcrA operon in Porphyromonas gingivalis. It encodes for a protein of unknown function upregulated under hydrogen peroxide (H2O2)-induced oxidative stress. Bioinformatic analysis shows that PG1037 has a zinc-finger motif, two peroxidase motifs, and one cytidylate kinase domain. The aim of this study is to characterize further the role of the PG1037 recombinant protein in the unique 8-oxoG repair system in P. gingivalis. PG1037 recombinant proteins with deletions in the zinc-finger or peroxidase motifs were created. Electrophoretic mobility shift assays were used to evaluate the ability of the recombinant proteins to bind 8-oxoG-containing oligonucleotides. Zinc binding, peroxidase, and Fenton reaction assays were used to assess the functional roles of the rPG1037 protein. A bacterial adenylate cyclase two-bride assay was used to identify the partner protein of PG1037 in the repair of 8-oxoG. The recombinant PG1037 (rPG1037) protein carrying an N-terminal His-tag demonstrated an ability to recognize and bind 8-oxoG-containing oligonucleotide. In contrast to the wild-type rPG1037 protein, the zinc-finger motif deletion resulted in the loss of zinc and 8-oxoG binding activities. A deletion of the peroxidase motif-1 showed a decrease in peroxidase activity. Using a bacterial adenylate cyclase two-hybrid system, there was no observed protein-protein interaction of PG1037 with UvrA (PG1036), PcrA (PG1038), or mismatch repair system proteins. Taken together, the results show that PG1037 is an important member of a novel mechanism that recognizes and repairs oxidative stress-induced DNA damage in P. gingivalis.

Rapid Detection of Methicillin Resistant Staphylococcus Aureus (MRSA) Using Electric-Double-Layer (EDL) Field-Effect Transistor (FET) Biosensors

According to the latest statistics and analysis by American researchers based on the data from 195 countries worldwide, it has been found that one person in every five people died due to sepsis. The annual death caused by sepsis reaches up to eleven million people, and the number of deaths from sepsis is even higher than those from cancer. We believe that the detection of MRSA is necessary.Staphylococcus aureus is the most common pathogen of sepsis. Methicillin-resistant Staphylococcus aureus (MRSA) or Multiple-resistant Staphylococcus aureus is a distinct strain of Staphylococcus aureus. It has mecA gene which codes for penicillin binding protein 2a (PBP 2A) that will remain active under beta lactams antibiotics. This gives bacterial the ability to grow under concentrations of beta-lactam antibiotics which leads to MRSA's resistance to almost all the different penicillin-type antibiotics including methicillin and other beta-lactamase-resistant penicillins.To detect MRSA in the shortest possible time, we adopt field-effect transistor (FET) biosensor for the real-time detection of binding reactions between DNA probe and cDNA. We select DNA probe sequence of mecA to be the aptamer and immobilize probe DNA which are complementary to the target cDNA on the sensor. This process allows DNA strands to attach to the gold surface. After that, we detect the binding reaction of DNA probe and cDNA by analyzing electrical response of field-effect transistor (FET). We can observe that the Id current changes after cDNA hybridizes with DNA probe.In conclusion, in our research results, Field-effect transistor (FET) biosensor can detect concentration ranging from 1fM to 1pM. In addition, we can achieve other advantages such as more portable, higher sensitivity, real-time monitoring capability, and lower detection limits. With this rapid detection procedure, we can do early diagnosis and treatment, thus improving the quality of human life. Keywords: FET, EDL, Aptamer, DNA, MRSA Figure 1

In Silico Identification and Molecular Mechanism of Novel Tyrosinase Inhibitory Peptides Derived from Nacre of Pinctada martensii.

Pearl and nacre powders have been valuable traditional Chinese medicines with whitening properties for thousands of years. We utilized a high-temperature and high-pressure method along with compound enzyme digestion to prepare the enzymatic hydrolysates of nacre powder of Pinctada martensii (NP-PMH). The peptides were identified using LC-MS/MS and screened through molecular docking and molecular dynamics simulations. The interactions between peptides and tyrosinase were elucidated through enzyme kinetics, circular dichroism spectropolarimetry, and isothermal titration calorimetry. Additionally, their inhibitory effects on B16F10 cells were explored. The results showed that a tyrosinase-inhibitory peptide (Ala-His-Tyr-Tyr-Asp, AHYYD) was identified, which inhibited tyrosinase with an IC50 value of 2.012 ± 0.088 mM. The results of the in vitro interactions showed that AHYYD exhibited a mixed-type inhibition of tyrosinase and also led to a more compact enzyme structure. The binding reactions of AHYYD with tyrosinase were spontaneous, leading to the formation of a new set of binding sites on the tyrosinase. The B16F10 cell-whitening assay revealed that AHYYD could reduce the melanin content of the cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that AHYYD could be widely used as a tyrosinase inhibitor in whitening foods and pharmaceuticals.

The improved purification technique for isolation of the novel CGTase from the alkaliphilc strain Caldalkalibacillus mannanilyticus IB-OR17-B1

Cyclodextrin-glucanotransferase (CGTase, EC 2.4.1.19) is a multifunctional enzyme that catalyzes many enzymatic reactions including cyclization, binding, disproportionation and hydrolysis reactions, playing an important role in the enzymatic synthesis of compounds that are widely used in agriculture, pharmaceuticals, food, chemical and biotechnology industries. The present research is aimed to optimize the purification protocol for the extracellular CGTase of alkaliphilc bacterial strain Caldalkalibacillus mannanilyticus IB-OR17-B1 guaranteeing the enzyme homogeneity and its high yield. The improved combination of ultrafiltration and corn-starch (5% w/v) affinity sorption techniques resulted to mild and rapid isolation of electrophoritically homogenic enzyme at 18 × increase of its specific activity and yield 56%. The developed two-step procedure instead the practiced tree-step one using commonly ion-exchange chromatography as final purification technique highly contributes in advance of cost-effectiveness for industrial production and isolation of valuable CGTases.

Binding affinity screening of polyphenolic compounds in Stachys affinis extract (SAE) for their potential antioxidant and anti-inflammatory effects

Free radical is a marker in various inflammatory diseases. The antioxidant effect protects us from this damage, which also plays an essential role in preventing inflammation. Inflammation protects the body from biological stimuli, and pro-inflammatory mediators are negatively affected in the immune system. Inflammation caused by LPS is an endotoxin found in the outer membrane of Gram-negative bacteria, which induces immune cells to produce inflammatory cytokines such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase. Based on this, the antioxidant and anti-inflammatory effects of plant extracts were investigated. First, the main phenolic compounds for the five peaks obtained from Stachys affinis extract (SAE) were identified. The antioxidant effect of each phenolic compound was confirmed through HPLC analysis before and after the competitive binding reaction between DPPH and the extract. Afterward, the anti-inflammatory effect of each phenolic compound was confirmed through competitive binding between COX2 and the extract in HPLC analysis. Lastly, the anti-inflammatory effect of SAE was confirmed through in vitro experiments and also confirmed in terms of structural binding through molecular docking. This study confirmed that phenolic compounds in SAE extract have potential antioxidant and anti-inflammatory effects, and may provide information for primary screening of medicinal plants.

Optical Label-Free Aptasensor Based on Weak Value Amplification for Real-Time and Ultrasensitive Detection of IgE.

Allergy is a prevalent disease, and the potential allergic population is expanding with industrialization and changes in people's living standards. Serum immunoglobulin E (IgE) level is one of the critical indicators for determining allergy. Here, we proposed a simple, real-time monitoring, low chip cost, label-free aptamer biosensing strategy based on weak value amplification (WVA) for the quantitative detection of IgE in serum samples, enabling early and accurate diagnosis of allergic or hypersensitive patients. The aptasensor combined an imaging weak measurement system with the high specificity of the aptamer for the marker IgE. By modifying the amino group at the 3-terminal end, the anti-IgE aptamers can attach to a dopamine-modified prism's surface and selectively recognize IgE in human serum. In the presence of IgE, a specific binding reaction occurred, resulting in a change in the refractive index of the reactive region's surface, manifested as a change in the light intensity of the camera acquired experimental images. As the concentration of IgE increased, the relative light intensity advanced sequentially. The WVA-aptasensing strategy achieved a wide detection range of 0.01 ng/mL to 2 μg/mL in phosphate buffered saline buffer, with the resolution as low as 4.3 pg/mL. IgE testing experiments in human serum have proved the feasibility of our methods in detecting complex samples. In addition, the method specifically recognized IgE without interference from other proteins. We believe that our proposed sensing strategy opens up new possibilities for ultrahigh sensitivity screening of IgE and can be expanded to detecting other biomolecules.

A Chemo-Mechanically Coupled DNA Origami Clamp Capable of Generating Robust Compression Forces.

DNA nanostructures have been utilized to study biological mechanical processes and construct artificial nanosystems. Many application scenarios necessitate nanodevices able to robustly generate large single molecular forces. However, most existing dynamic DNA nanostructures are triggered by probabilistic hybridization reactions between spatially separated DNA strands, which only non-deterministically generate relatively small compression forces (≈0.4 piconewtons (pN)). Here, an intercalator-triggered dynamic DNA origami nanostructure is developed, where large amounts of local binding reactions between intercalators and the nanostructure collectively lead to the robust generation of relatively large compression forces (≈11.2 pN). Biomolecular loads with different stiffnesses, 3, 4, and 6-helix DNA bundles are efficiently bent by the compression forces. This work provides a robust and powerful force-generation tool for building highly chemo-mechanically coupled molecular machines in synthetic nanosystems.

An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses

Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance therapeutics for blocking macrophage phagocytosis checkpoint inhibitors in solid tumors. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. By simultaneously blocking CD47 and CD24 signaling, PAC-SABI enhances the phagocytic ability of macrophages in vitro and in vivo, promoting anti-tumor responses in breast and pancreatic cancer mouse models. Moreover, building on the foundation of PAC-SABI-induced macrophage repolarization and increased CD8+ T cell tumor infiltration, sequential anti-PD-1 therapy further suppresses 4T1 tumor progression, prolonging survival rate. The in vivo construction of PAC-SABI-based nano-architectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency.

Comprehending the inhibition mechanism of indole-based bis-acylhydrazone compounds on α-glucosidase: Spectral and theoretical approaches

Indole-based bis-acylhydrazone compounds can inhibit the activity of α-glucosidase and control the concentration of blood glucose. In this paper, the characteristics of three indole-based bis-acylhydrazone compounds with different inhibitory activities of α-glucosidase as well as the interaction with α-glucosidase were studied by experiments and computational simulation techniques. Enzyme kinetic and spectral experiments showed that the indole-based bis-acylhydrazone compounds were able to inhibit enzyme activity through mixed inhibition dominated by competitive inhibition, and during the binding reaction, indole-based bis-acylhydrazone compounds can quench the intrinsic fluorescence of α-glucosidase through static quenching and an aggregation of the indole-based bis-acylhydrazone with α-glucosidase produces a stable complex with a molar ratio of 1:1, and the combination of indole-based bis-acylhydrazone compounds could lead to slight change in the conformation of α-glucosidase. The theoretical simulation demonstrated that the stability of the complex systems was positively correlated with the inhibitory activity of indole-based bis-acylhydrazone compounds, and the indole-based bis-acylhydrazone compounds occupied the active site in the multi-ligand system, resulting in a significant decrease in the binding ability of starch to active amino acids. These results suggested that indole-based bis-acylhydrazone compound was expected to be a new type of α-glucosidase inhibitor.

Understanding Microscopic Interactions in Binding Reactions: The pH Titration of EDTA

ABSTRACT Students beginning the study of biochemistry or biophysics at the undergraduate or even early graduate level are often overwhelmed by the complexity of the systems and the nomenclature. By comparison, chemical systems appear simple, as students can more easily relate to introductory chemistry courses, where the molecules are smaller and bind yet smaller ions. This allows students to write the structure of the entire molecule on a piece of paper and see exactly to which functional groups an ion, such as a proton (H+) in the simplest case, binds. Yet, concepts that are fundamental in biochemical macromolecules, namely proteins, can perfectly well be taught at the undergraduate or beginning graduate level, and probably be more easily understood, by using simpler, familiar chemical examples. The concept of interacting binding sites, which is at the root of cooperativity in protein binding reactions and conformational changes, is already present in simple molecules, such as ethylenediaminetetraacetic acid (EDTA). In this article, we show how to teach these topics by using the idea of the partition function, rather than a formal algebraic approach, to treat the binding of protons to EDTA. Profound concepts, such as that of interacting sites, appear naturally in a small molecule, where the origin can be easily ascribed, in this case, mainly to electrostatic interactions. Equipped with this understanding and this approach, students will be able to tackle more complicated biochemical systems, in which the molecules are larger, but the concepts are the same.

Isolated auto-citrullinated regions of PADI4 associate to the intact protein without altering their disordered conformation

PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions – i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.