Isothermal Titration Calorimetry Analysis Research Articles

Structure-based drug design of DNA minor groove binders and evaluation of their antibacterial and anticancer properties

Antimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.

5-methyl-salicylaldoxime based ionic liquids for non-destructive separation of protein and cadmium

Confronting with explosive cadmium contaminations to environment, non-destructive separation of protein with cadmium for protecting food safety is hardpressed. Ionic liquids (ILs) are benign bio-compatible stabilizer of proteins with considerable metal affinity, while their application in separation of cadmium from protein is limited owing to their comparative metal affinities with protein. Accordingly, we firstly synthesize 1-methyl-3 (salicylaldoxime-5-methyl) imidazole hexafluorophosphate ([Salenmim][PF6]) to enhance cadmium extraction with hydroxyl and oxime groups as ligands of cadmium. Structure of [Salenmim][PF6] is confirmed by 1H NMR, 13C NMR, 31P NMR, 19F NMR, FT-IR, Raman spectra, and theoretical simulations. [Salenmim][PF6] can remove 85.31 % of Cd2+ from bovine serum albumin (BSA) at pH of 2.0. X-ray photoelectron spectroscopy (XPS) of BSA and [Salenmim][PF6] before and after Cd2+ extraction illustrate that Cd2+ generally bonds with C = N and O-H of [Salenmim][PF6] to achieve detachment of Cd2+ from BSA. Molecular docking of BSA and Cd2+ indicates that 4 amino acids residues listed as ASP561, ASP562, LYS563, and GLU564 coordinate with Cd2+ with coordinate number close to 1, further confirmed as 1.48 by isothermal titration calorimetry (ITC) analysis. DFT calculations of [Salenmim][PF6] with Cd2+ demonstrate that energy gap between HOMO orbital localized at benzene ring is 1.6860 eV higher than LUMO orbital, faciliating formation of [Salenmim][PF6]-Cd2+ complex. Finally, there are no changes in secondary and tertiary structure of BSA and no ILs residual observed after [Salenmim][PF6] assisted Cd2+ extraction. This work firstly reports a method for synthesis of [Salenmim][PF6] with high affinity to cadmium and systemically reveals how do structure of [Salenmim][PF6] enhance heavy metal removal from proteins, providing a possible strategy for heavy metal extraction from proteins.

Structural characterization and mechanisms of macrophage immunomodulatory activity of a novel polysaccharide with a galactose backbone from the processed Polygonati Rhizoma

A purified polysaccharide with a galactose backbone (SPR-1, Mw 3,622 Da) was isolated from processed Polygonati Rhizoma with black beans (PRWB) and characterized its chemical properties. The backbone of SPR-1 consisted of [(4)-β-D-Galp-(1]9→4,6)-β-D-Galp-(1→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→4)-α-D-Glcp-(1→4,6)-α-D-Glcp-(1→4)-α/β-D-Glcp, with a branch chain of R1: β-D-Galp-(1→3)-β-D-Galp-(1→ connected to the →4,6)-β-D-Galp-(1→ via O-6, and a branch chain of R2: α-D-Glcp-(1→6)-α-D-Glcp-(1→ connected to the →4,6)-α-D-Glcp-(1→ via O-6. Immunomodulatory assays showed that the SPR-1 significantly activated macrophages, and increased secretion of NO and cytokines (i.e., IL-1β and TNF-α), as well as promoted the phagocytic activities of cells. Furthermore, isothermal titration calorimetry (ITC) analysis and molecular docking results indicated high-affinity binding between SPR-1 and MD2 with the equilibrium dissociation constant (KD) of 18.8 μM. It was suggested that SPR-1 activated the immune response through Toll-like receptor 4 (TLR4) signaling and downstream responses. Our research demonstrated that the SPR-1 has a promising candidate from PRWB for the TLR4 agonist to induce immune response, and also provided an easily accessible way that can be used for PR deep processing.

Isothermal titration calorimetry analysis of the binding between the maltodextrin binding protein malE of Staphylococcus aureus with maltodextrins of various lengths

Staphylococcus aureus (S. aureus), a Gram-positive bacterium, causes a wide range of infections, and diagnosis at an early stage is challenging. Targeting the maltodextrin transporter has emerged as a promising strategy for imaging bacteria and has been able to image a wide range of bacteria including S. aureus. However, little is known about the maltodextrin transporter in S. aureus, and this prevents new S. aureus specific ligands for the maltodextrin transporter from being developed. In Gram-positive bacteria, including S. aureus, the first step of maltodextrin transport is the binding of the maltodextrin-binding protein malE to maltodextrins. Thus, understanding the binding affinity and characteristics of malE from S. aureus is important to developing efficient maltodextrin-based imaging probes. We evaluated the affinity of malE of S. aureus to maltodextrins of various lengths. MalE of S. aureus (SAmalE) was expressed in E. coli BL21(DE3) and purified by Ni-NTA resin. The affinities of SAmalE to maltodextrins were evaluated with isothermal titration calorimetry. SAmalE has low affinity to maltose but binds to maltotriose and longer maltodextrins up to maltoheptaose with affinities up to Ka = 9.02 ± 0.49 × 105 M−1. SAmalE binding to maltotriose-maltoheptaose was exothermic and fit a single-binding site model. The van't Hoff enthalpy in the binding reaction of SAmalE with maltotriose was 9.9 ± 1.3 kcal/mol, and the highest affinity of SAmalE was observed with maltotetraose with Ka = 9.02 ± 0.49 × 105 M−1. In the plot of ΔH-T*ΔS, the of Enthalpy−Entropy Compensation effect was observed in binding reaction of SAmalE to maltodextrins. Acarbose and maltotetraiol bind with SAmalE indicating that SAmalE is tolerant of modifications on both the reducing and non-reducing ends of maltodextrins. Our results show that unlike ECmalE and similar to the maltodextrin binding protein of Streptococci, SAmalE primarily binds to maltodextrins via hydrogen bonds. This is distinct from the maltodextrin binding protein of Streptococci, SAmalE that binds to maltotetraiol with high affinity. Understanding the binding characteristics and tolerance to maltodextrins modifications by maltodextrin binding proteins will hopefully provide the basis for developing bacterial species-specific maltodextrin-based imaging probes.

In silico identification and molecular mechanism of novel egg white-derived tyrosinase inhibitory peptides

This study aimed to identify novel tyrosinase inhibitory peptides from egg white proteins including ovalbumin, ovotransferrin and ovomucoid. Peptides GDVA and DEK were identified using computer-aided virtual screening and in vitro tyrosinase inhibition experiments. Especially, peptide DEK exhibited the strongest tyrosinase inhibitory capacity with IC50 value of 0.205 ± 0.07 mmol L−1. Molecular docking and isothermal titration calorimetry analysis (ITC) were performed to explore the molecular mechanism. The results revealed that two peptides interacted with key residues of tyrosinase mainly by hydrogen bonding and hydrophobic interaction. The binding reactions of these peptides with tyrosinase were spontaneous, endothermic and entropy driven. Furthermore, the structure of two peptide-tyrosinase complexes was compact and stable during the molecular dynamic simulation. These findings indicated that egg white-derived peptide DEK had potential as tyrosinase inhibitory peptide.

Zwitterionic Polysulfobetaine Inhibits Cancer Cell Migration Owing to Actin Cytoskeleton Dynamics.

Cell migration is an essential dynamic process for most living cells, mainly driven by the reorganization of actin cytoskeleton. To control actin dynamics, a molecular architecture that can serve as a nucleator has been designed by polymerizing sulfobetaine methacrylate. The synthesized zwitterionic polymer, poly(sulfobetaine methacrylate) (PZI), effectively nucleates the polymerization process of G-actin and substantially accelerates the rate of polymerization. Isothermal titration calorimetry (ITC) and bioinformatics analysis indicated binding between PZI and monomeric G-actin. Thus, in vitro actin dynamics was studied by dynamic light scattering (DLS), pyrene-actin polymerization assay, and total internal reflection fluorescence microscopy (TIRFM). Furthermore, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore-containing monomeric unit was incorporated into the sulfobetaine zwitterionic architecture to visualize the effect of polymer in the cellular environment. The BODIPY-containing zwitterionic sulfobetaine polymer (PZI-F) successfully penetrated the cell and remained in the lysosome with minimal cytotoxicity. Confocal microscopy revealed the influence of this polymer on the cellular actin cytoskeleton dynamics. The PZI-F polymer was successfully able to inhibit the collective migration of the human cervical cancer cell line (HeLa cell) and breast cancer cell line (MDA-MB-231 cell), as confirmed by a wound healing assay. Therefore, polyzwitterionic sulfobetaine could be explored as an inhibitor of cancer cell migration.

ITC analysis of polydisperse systems: Unravelling the impact of sample heterogeneity

Binding interactions often involve heterogeneous samples displaying a distribution of binding sites that vary in affinity and binding enthalpy. Examples include biological samples like proteins and chemically produced samples like modified cyclodextrins. Experimental studies often ignore sample heterogeneity and treat the system as an interaction of two homogeneous species, i.e. a chemically well-defined ligand binding to one type of site. The present study explores, by simulations and experiments, the impact of heterogeneity in isothermal titration calorimetry (ITC) setups where one of the binding components is heterogeneous. It is found that the standard single-site model, based on the assumption of two homogeneous binding components, provides excellent fits to simulated ITC data when the binding free energy is normally distributed and all sites have similar binding enthalpies. In such cases, heterogeneity can easily go undetected but leads to underestimated binding constants. Heterogeneity in the binding enthalpy is a bigger problem and may result in enthalpograms of increased complexity that are likely to be misinterpreted as two-site binding or other complex binding models. Finally, it is shown that heterogeneity can account for previously observed experimental anomalies. All simulations are accessible in Google Colab for readers to experiment with the simulation parameters.

Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50) values ranging from 1.42 to 32.7 μM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC50) values of 21.73 and 31.19 μM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.

HIF-1α inhibition by MO-2097, a novel chiral-free benzofuran targeting hnRNPA2B1

IntroductionHypoxia-inducible factor 1 (HIF-1) is a transcriptional activator mediating adaptive responses to hypoxia. It is up-regulated in the tumor microenvironment and recognized as an effective anticancer drug target. Previously, we discovered that the natural compound moracin-O and its synthetic derivative MO-460 inhibited HIF-1α via hnRNPA2B1. ObjectivesThis study aimed to develop novel HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural products moracins-O and P. MethodsIn an ongoing search for novel HIF-1 inhibitors, a series of nature-inspired benzofurans with modifications on the chiral rings of moracins-O and P were synthesized. They showed improved chemical tractability and were evaluated for their inhibitory activity on HIF-1α accumulation under hypoxic conditions in HeLa CCL2 cells. The most potent derivative's chemical-based toxicities, binding affinities, and in vivo anti-tumorigenic effects were evaluated. Further, we examined whether our compound, MO-2097, exhibited anticancer effects in three-dimensional cultured organoids. ResultsHerein, we identified a novel synthetic chiral-free compound, MO-2097, with reduced structural complexity and increased efficiency. MO-2097 exhibited inhibitory effects on hypoxia-induced HIF-1α accumulation in HeLa CCL2 cells via inhibition of hnRNPA2B1 protein, whose binding affinities were confirmed by isothermal titration calorimetry analysis. In addition, MO-2097 demonstrated in vivo efficacy and biocompatibility in a BALB/c mice xenograft model. The immunohistochemistry staining of MO-2097-treated tissues showed decreased expression of HIF-1α and increased levels of apoptosis marker cleaved caspase 3, confirming in vivo efficacy. Furthermore, we confirmed that MO-2097 works effectively in cancer patient-based organoid models. ConclusionMO-2097 represents a promising new generation of chemotherapeutic agents targeting HIF-1α inhibition via hnRNPA2B1, requiring further investigation.

Enhanced Ca2+ binding to EF-hands through phosphorylation of conserved serine residues activates MpRBOHB and chitin-triggered ROS production.

NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca2+ binding and phosphorylation by various protein kinases, a mechanism linking Ca2+ binding and phosphorylation in the activity regulation remained elusive. Chitin-triggered ROS production required cytosolic Ca2+ elevation and Ca2+ binding to MpRBOHB in a liverwort Marchantia polymorpha. Heterologous expression analysis of truncated variants revealed that a segment of the N-terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF-hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta. Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca2+ binding affinity of MpRBOHB, while Ca2+ binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca2+ -dependent activation of MpRBOHB, emphasizing the pivotal role of Ca2+ binding in mediating the Ca2+ and phosphorylation-driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.

Importance of aspartate 4 in the Mg2+ dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

Carbazole diester strapped calix[4]pyrrole: Synthesis, anion binding and self-assembly based on ion pair recognition

A new carbazole strapped calix[4]pyrrole 1 has been synthesized and ion binding properties was investigated using 1H NMR, isothermal titration calorimetry and single crystal X-ray diffraction analyses. Compared to the parent octamethyl calix[4]pyrrole 2, receptor 1 showed enhanced binding towards halide anions. Solid state structural analysis revealed that receptor 1 is capable of binding various halide salts showing different binding modes depending on the bound ion pairs. Interestingly, complexes of tetrabutyammonium (TBA+), tetraethylammonium (TEA+) and tetramethylammonium (TMA+) salts of chloride anion displayed three distinctly different binding geometries in the solid state. As a result, the linear supramolecular polymers seen in the crystal lattices are also dramatically different for these three chloride complexes of 1. Combination of 1H NMR and solid state studies revealed that binding of anion in one condition do not translates to another completely. Receptor 1 also found to form 1/1 complexes with CsCl, CsBr and CsI salts and 1D coordination polymer chains are stabilized in the solid state. Overall, construction of four different 1D linear chains are described for chloride anion based on ion pair recognition. In addition, the solid state structure of ion pair complex with CsI salt is unprecedented in the calix[4]pyrrole chemistry. Moreover, under solid-to-liquid extraction condition, receptor 1 is able to solubilise CsF and CsCl ion pairs in chloroform in which these salts are otherwise insoluble.

Conformational entropy of hyaluronic acid contributes to taste enhancement

Natural taste/flavor enhancers are essential ingredients that could potentially address condiments overconsumption. For the first time, we report that hyaluronic acid (HA) could modulate taste perception, governed by the dynamic interactions among taste compounds, mucin, and HA. Various conformations of HA impact taste perception. The high molecular weight (Mw) of 1090 kDa HA inhibits the sense of taste due to its increased viscosity, which hinders the penetration of Na+ into the mucin layer. HA with low and medium Mw (100 kDa, 400 kDa) could enhance taste perception. Isothermal titration calorimetry analysis confirms the stronger binding between mucin and HA. The intensity of their interaction increases as the Mw of HA increases from 8 kDa to 400 kDa. Quartz crystal microbalance with dissipation characterization further indicates that the rigid conformation of 100 kDa HA facilitates the binding of Na+ with taste receptors, thereby enhancing taste perception. The flexible conformation of 400 kDa HA may conceal the taste receptor cells, reducing taste enhancement. Our work advances the understanding of conformational entropy of natural mucoadhesion and mucopenetration polymers, which lays the foundation for their potential use as taste enhancers.

Quantifying cooperative multisite binding in the hub protein LC8 through Bayesian inference.

Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. In this study, we primarily examine the essential hub protein LC8, a small dimer that binds disordered regions of 100+ client proteins in two symmetrical grooves at the dimer interface. Mechanistic details of LC8 binding have remained elusive, hampered in part by ITC data analyses employing simple models that treat bivalent binding as a single event with a single binding affinity. We build on existing Bayesian ITC approaches to quantify thermodynamic parameters for multi-site binding interactions impacted by significant uncertainty in protein concentration. Using a two-site binding model, we identify positive cooperativity with high confidence for LC8 binding to multiple client peptides. In contrast, application of an identical model to the two-site binding between the coiled-coil NudE dimer and the intermediate chain of dynein reveals little evidence of cooperativity. We propose that cooperativity in the LC8 system drives the formation of saturated induced-dimer structures, the functional units of most LC8 complexes. In addition to these system-specific findings, our work advances general ITC analysis in two ways. First, we describe a previously unrecognized mathematical ambiguity in concentrations in standard binding models and clarify how it impacts the precision with which binding parameters are determinable in cases of high uncertainty in analyte concentrations. Second, building on observations in the LC8 system, we develop a system-agnostic heat map of practical parameter identifiability calculated from synthetic data which demonstrates that the ability to determine microscopic binding parameters is strongly dependent on both the parameters themselves and experimental conditions. The work serves as a foundation for determination of multi-step binding interactions, and we outline best practices for Bayesian analysis of ITC experiments.

Discovery of anti-allergic components in Guomingkang Formula using sensitive HEMT biochips coupled with in vitro and in vivo validation

BackgroundAllergic rhinitis (AR) is a prevalent allergic disease, which seriously affects the sufferers’ life quality and increases the socioeconomic burden. Guominkang (GMK), a well-known prescription for AR treatment, showed satisfactory effects; while its anti-allergic components remain to be disclosed. AlGaN/GaN HEMT biochip is more sensitive and cost-effective than other binding equipments, indicating its great potential for screening of active ingredients from herbal medicines. MethodsAR mouse models were first established to test the anti-allergic effect of GMK and discover the ingredients absorbed into blood by ultra-high performance liquid chromatography-mass spectra (UHPLC-MS). Then, novel Syk/Lyn/Fyn-functionalized high electron mobility transistor (HEMT) biochips with high sensitivity and specificity were constructed and applied to screen the active components. Finally, the results from HEMT biochips screening were validated via in silico (molecular docking and molecular dynamics simulation), in vitro (RBL-2H3 cells), and in vivo (PCA mice model) assays. ResultsGMK showed a potent therapeutic effect on AR mice, and fifteen components were identified from the medicated plasma. Furthermore, hamaudol was firstly found to selectively inhibit the Syk and Lyn, and emodin was to selectively inhibit Lyn, which were further confirmed by isothermal titration calorimetry, molecular docking, and molecular dynamics simulation analyses. Suppression of the activation of FcεRI-MAPK signals might be the possible mechanism of the anti-allergic effect of hamaudol. ConclusionsThe targets of emodin and hamaudol were discovered by HEMT biochips for the first time. This study provided a novel and effective strategy to discover active components in a complex herbal formula by using AlGaN/GaN HEMT biochips.

Investigation of bovine β-lactoglobulin-procyanidin complexes interactions and its utilization in O/W emulsion

Procyanidins are bioactive polyphenols that have a strong affinity to proteins. Beta-lactoglobulin (BLG) is widely used as an emulsifier in the food and other industries. This study evaluated the interaction between BLG and A-type procyanidin dimer (PA2) using the spectroscopic, thermodynamic, and molecular simulation. PA2 decreased the transmissivity and quenched the intrinsic fluorescence of BLG, suggesting that the two molecules formed a complex. The binding of PA2 reduced the surface hydrophobicity and altered the conformation of BLG with increasing the random coil regions. Thermodynamic and isothermal titration calorimetry analyses suggested that the main driving force of PA2-BLG interaction was hydrophobic attraction. Molecular docking simulations were used to identify the main interaction sites and forces in the BLG-PA2 complexes, which again indicated that hydrophobic interactions dominated. In addition, the influence of PA2 on the ability of BLG to form and stabilize O/W emulsions was analyzed. Emulsions formulated using BLG-PA2 complexes contained relatively small droplets (D4,3 ≈ 0.7 μm) and high surface potentials (absolute value >50 mV). Compared to BLG alone, BLG-PA2 complexes improved the storage stability of the emulsions. This study provides valuable new insights into the formation, properties, and application of protein-polyphenol complexes as functional ingredients in foods.

Mechanism of interaction between urolithin A and α-glucosidase: Analysis by inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry and molecular docking

In this study, the mechanism of interaction between urolithin A, a gut metabolite of ellagitannins, and α-glucosidase was characterized by inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry, and molecular docking. Urolithin A exhibited potential reversible inhibitory activity for α-glucosidase in an uncompetitive manner with an IC50 value of (28.03 ± 0.59) μM. The results of fluorescence titration and isothermal titration calorimetry analysis showed that urolithin A statically quenched the endogenous fluorescence of α-glucosidase, which was a spontaneous exothermic process, mainly driven by hydrogen bond and/or van der Waals force. Synchronous fluorescence spectroscopy revealed that urolithin A increased the polarity of tryptophan microenvironment and the hydrophobicity of tyrosine microenvironment. The combination of urolithin A with acarbose, a competitive α-glucosidase inhibitor, showed an additive inhibitory effect. Furthermore, molecular docking showed that urolithin A formed hydrogen bonds with key residues Arg269, Thr273, and His258, which are the sites outside the active center of α-glucosidase. These findings could highlight the value of ellagitannin rich diet as an antihyperglycemic food in the prevention of postprandial hyperglycemia, and indicate that urolithin A may be a promising lead compound of α-glucosidase inhibitor.

Crystal structures and identification of novel Cd2+-specific DNA aptamer.

Cadmium (Cd) is one of the most toxic heavy metals. Exposure to Cd can impair the functions of the kidney, respiratory system, reproductive system and skeletal system. Cd2+-binding aptamers have been extensively utilized in the development of Cd2+-detecting devices; however, the underlying mechanisms remain elusive. This study reports four Cd2+-bound DNA aptamer structures, representing the only Cd2+-specific aptamer structures available to date. In all the structures, the Cd2+-binding loop (CBL-loop) adopts a compact, double-twisted conformation and the Cd2+ ion is mainly coordinated with the G9, C12 and G16 nucleotides. Moreover, T11 and A15 within the CBL-loop form one regular Watson-Crick pair and stabilize the conformation of G9. The conformation of G16 is stabilized by the G8-C18 pair of the stem. By folding and/or stabilizing the CBL-loop, the other four nucleotides of the CBL-loop also play important roles in Cd2+binding. Similarly to the native sequence, crystal structures, circular dichroism spectrum and isothermal titration calorimetry analysis confirm that several variants of the aptamer can recognize Cd2+. This study not only reveals the underlying basis for the binding of Cd2+ ions with the aptamer, but also extends the sequence for the construction of novel metal-DNA complex.

The crystalline structure transition and hydrogen bonds shift determining enhanced enzymatic digestibility of cellulose treated by ultrasonication

Global energy issue raised the necessity to develop second-generation biofuels, and biorefinery of cellulosic biomass becomes a promising solution. Various pretreatments were used to overcome the cellulose nature of recalcitrance and improve the enzymatic digestibility, but the lack of mechanism understanding hindered the development of efficient and cost-effective technologies of cellulose utilization. Using structure-based analysis, we demonstrate that the improved hydrolysis efficiency caused by ultrasonication was ascribed to the changed cellulose properties rather than the increased dissolubility. Further, isothermal titration calorimetry (ITC) analysis suggested that enzymatic digestion of cellulose is an entropically favored reaction driven by hydrophobic forces other than an enthalpically favored reaction. The changes in cellulose properties and thermodynamic paramenters due to ultrasonication accounted for the improved accessibility. Ultrasonication-treated cellulose showed porous, rough and disordered morphology, accompanying with the loss of crystalline structure. Despite the unaffected unit cell structure, ultrasonication expanded the crystalline lattice by increasing grain sizes and average cross-sectional area, resulting in the transformation from cellulose I to cellulose II, with the decreased crystallinity, better hydrophilicity and increased enzymatic bioaccessibility. Furthermore, FTIR combined with two-dimensional correlation spectroscopy (2D-CoS) verified that the sequential shift of hydroxyl group and intramolecular/intermolecular hydrogen bonds, the functional groups governing cellulose crystal structure and stability, accounted for the ultrasonication-induced transition of cellulose crystalline structure. This study provides a comprehensive picture of cellulose structure and property response caused by mechanistic treatments and will open up avenues to develop novel pretreatments for efficient utilization.

Effect of sodium alginate on the yogurt stability was dependent on the thickening effect and interaction between casein micelles and sodium alginate

The effect of sodium alginate (SA) on the yogurt stability and the related mechanisms were investigated. It was found that low-concentration SA (≤0.2 %) increased the yogurt stability, while high-concentration SA (≥0.3 %) decreased the yogurt stability. Sodium alginate increased the viscosity and viscoelasticity of yogurt and this effect was positively correlated with its concentration, suggesting that SA worked as the thickening agent in yogurt. However, addition of ≥0.3 % SA damaged the yogurt gel. These results suggested that interaction between milk protein and SA might play an important role in the yogurt stability besides the thickening effect. Addition of ≤0.2 % SA did not change the particle size of casein micelles. However, addition of ≥0.3 % SA induced aggregation of casein micelles and increased the size. And the aggregated casein micelles precipitated after 3 h storage. Isothermal titration calorimetry analysis showed that casein micelles and SA were thermodynamically incompatible. These results suggested that the interaction between casein micelles and SA induced aggregation and precipitation of casein micelles, which was critical in the destabilization of yogurt. In conclusion, the effect of SA on the yogurt stability was dependent on the thickening effect and the interaction between casein micelles and SA.