Isothermal Titration Calorimetry Analysis Research Articles

Evaluation of Activity of Sesquiterpene Lactones and Chicory Extracts as Acetylcholinesterase Inhibitors Assayed in Calorimetric and Docking Simulation Studies.

The aim of the study was to explain the effects of sesquiterpene lactones (SLs) from chicory (Cichorium intybus L.) root extracts as inhibitors of acetylcholinesterase (AChE) at the molecular level and to determine the inhibition of AChE activity by specific SLs (lactucin and lactucopicrin) and different chicory extracts. The obtained SLs-rich extracts were purified by the countercurrent partition chromatography (CPC) technique. AChE inhibitors were analyzed using two models: isothermal titration calorimetry (ITC) and docking simulation. The results of ITC analysis of the enzyme and the ligands’ complexation showed strong interactions of SLs as well as extracts from chicory with AChE. In a test of enzyme activity inhibition after introducing acetylcholine into the model system with SL, a stronger ability to inhibit the hydrolysis of the neurotransmitter was observed for lactucopicrin, which is one of the dominant SLs in chicory. The inhibition of enzyme activity was more efficient in the case of extracts, containing different enzyme ligands, exhibiting complementary patterns of binding the AChE active site. The study showed the high potential of using chicory to decrease the symptoms of Alzheimer’s disease.

The binding of secondary chromophore for thermally stable rhodopsin makes more stable with temperature

AbstractMicrobial rhodopsin is a transmembrane protein that functions with a chromophore and is regulated by light. In nature, regulation through the retinal chromophore plays an important role in physiological phenomena. However, living organisms in soil, sea, and freshwater synthesize carotenoids preferentially over retinal in the biosynthetic pathway. Evolution has extended the energy conversion photosystem with additional pigments that act as antennae. Previously, Gloeobacter rhodopsin and xanthorhodopsin have been reported to form secondary chromophores with carotenoids. In this study, we report that a thermophilic rhodopsin (TR) and Tara76 rhodopsin, the latter of which is classified as a blue light‐absorbing proteorhodopsin, can form secondary chromophores with canthaxanthin (CAN). Tara76 rhodopsin and TR were found to exhibit high thermal stabilities and photophysical properties following their interaction with CAN. Isothermal titration calorimetry analysis, spectral shift measurements, and exciton analysis were used to examine the interactions of these rhodopsins with CAN. It was found that these interactions increased the stability toward temperature and pH through highly efficient chromophore formation, in addition to rapidly recruiting the retinal at a rate approximately twice as high as that obtained in the absence of CAN.

Discovery of Novel α-Methylene-γ-Butyrolactone Derivatives Containing Vanillin Moieties as Antiviral and Antifungal Agents.

On the basis of the structure of nicotlactone A (L1), a series of novel α-methylene-γ-butyrolactone derivatives B1-B43 were designed and synthesized by structure simplification and active fragment replacement strategies, and their antiviral and antifungal activities were evaluated. The bioassay studies indicated that many target compounds possessed good to excellent antiviral activity against tobacco mosaic virus (TMV) and some of these compounds exhibited specific antifungal activities against Valsa mali and Fusarium graminearum. Compound B32 exhibited the best anti-TMV activity (inactivation effect, 88.9%; protection effect, 65.8%; curative effect, 52.8%) in vivo at 500 mg/L, which is significantly higher than that of commercial virucides ribavirin and ningnanmycin. The inhibition effect of compound B32 was also visualized by the inoculation test using green fluorescent protein (GFP)-labeled TMV. The preliminary antiviral mechanism of compound B32 was investigated. Transmission electron microscopy (TEM) showed that compound B32 could destroy the integrity of virus particles. Then, molecular docking and isothermal titration calorimetry (ITC) analysis further demonstrated that compound B32 exhibited a strong binding affinity to the TMV coat protein with a dissociation constant (Kd) of 3.06 μM, superior to ribavirin. Thus, we deduced that compound B32 may interfere with the self-assembly of TMV particles by binding TMV coat protein (CP). In addition, compound B28 showed good in vitro activity against F. graminearum with an inhibition rate of 90.9% at 50 mg/L, which was greater than that of fluxapyroxad (59.1%) but lower than that of the commercial fungicide carbendazim (96.8%). The present study provides support for the application of these α-methylene-γ-butyrolactone derivatives as novel antiviral and antifungal agents in crop protection.

Effect of pH on the conformational structure of cytochrome c and subsequent enzymatic cross-linking catalyzed by laccase

BackgroundThe aim of the present study was to investigate the effect of substrate conformational structure changes on the laccase-induced protein cross-linking. The effects of laccase amount, pH, and ferulic acid (FA) on the enzymatic cross-linking of substrate, Cyt C, were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. High-performance size exclusion chromatography, laser particle size analysis and isothermal titration calorimetry (ITC) were also applied to investigate the cross-linking product and enthalpy changes. Structural changes of Cyt C at different pH values were analyzed by ultraviolet–visible (UV–vis), fluorescence, and circular dichroism (CD) measurements. ResultsComplete cross-linking, partial cross-linking, minute cross-linking, and no cross-linking occurred at pH 2.0, 4.0, 6.0, and 8.0, respectively. ITC analysis demonstrated that the enzymatic cross-linking of Cyt C was an endothermic process. The UV–vis, fluorescence, and CD measurements exhibited that the tertiary structure of Cyt C was disrupted, and part of the α-helical polypeptide region unfolded at pH 2.0. The structural flexibilities decreased, and the tertiary structure of Cyt C became increasingly compact with the increase in pH values from 4.0 to 8.0. The gradual changes in the structure of Cyt C at different pH values were in accordance with the cross-linking results of Cyt C catalyzed by laccase. ConclusionsThe results demonstrated that minute structure changes of substrate had a remarkable effect on the laccase-induced cross-linking. The findings promote the understanding of the substrate requirement of laccase in protein cross-linking and are instructive for the modulation of laccase-induced protein cross-linking.How to cite: Li D-X, Qi Z-Y, Liu J-Y, et al. Effect of pH on the conformational structure of cytochrome c and subsequent enzymatic cross-linking catalyzed by laccase. Electron J Biotechnol 2022;60. https://doi.org/10.1016/j.ejbt.2022.07.002.

Hemopexin as a Potential Binding Partner of Arginine-Rich Cell-Penetrating Peptides in Serum.

Serum proteins affect the in vivo fate and cellular uptake of arginine-rich cell-penetrating peptides (CPPs) and drugs delivered by CPPs. Although the binding of CPPs to serum proteins may possibly reduce their cellular uptake to some extent, it may also prolong their circulation half-life in vivo. We aimed to identify novel binding proteins of arginine-rich CPPs in serum to better understand their in vivo fate and develop more sophisticated drug delivery systems using CPPs. Isothermal titration calorimetry analysis suggests that albumin, the most abundant protein in serum, binds to d-forms of oligoarginine; however, the dissociation constants are several tens of a micromolar. Candidate proteins with the potential of binding to arginine-rich CPPs in serum were then explored using nondenaturing polyacrylamide gel electrophoresis followed by mass spectrometry analysis. Studies using fluorescence correlation spectroscopy determined hemopexin as a potential binding partner of d-forms of arginine-rich CPPs, including d-octaarginine (r 8) and the d-form of the peptide, corresponding to HIV-1 Rev (34-50), with dissociation constants of submicromolar to micromolar range. Using flow cytometry and a split-luciferase-based system, the promotion effect of hemopexin on the total cellular uptake and cytosolic localization of cargos conjugated with these CPPs was confirmed. Therefore, this study elucidated hemopexin as a potential binding partner of d-arginine-rich CPPs that may affect their in vivo fate and cellular uptake.

Structural requirements and interaction mechanisms of ACE inhibitory peptides: molecular simulation and thermodynamics studies on LAPYK and its modified peptides

The understanding of the structural requirements and the intermolecular-interaction mechanism are important for discovering potent angiotensin-converting enzyme (ACE) inhibitory peptides. In this study, we modified an egg-white derived peptide, LAPYK, using the amino acids with different properties to produce the LAPYK-modified peptides. The ACE inhibitory activities of the modified peptides were determined to explore the structural requirements of ACE inhibitory peptides (ACEIPs). Molecular simulation and isothermal titration calorimetry analysis were used to investigate interactions between the peptides and ACE. We found that hydrophobicity and the amino acids with ring structures were beneficial for the ACE inhibitory activities of the peptides. The results of the molecular mechanics poisson boltzmann surface area (MMPBSA) binding free energy calculations indicated that the polar solvation free energy (ΔGpolar) of the charged peptides (LAPYK, LAPYE) were unfavorable for binding to ACE. On the other hand, the results of isothermal titration calorimetry analyses suggested that the enthalpy-driven ACE-peptide interactions were more favorable than the entropy-driven ACE-peptide interaction counterparts.

Inhibiting Cyclin-Dependent Kinase 6 by Taurine: Implications in Anticancer Therapeutics.

Cyclin-dependent kinase 6 (CDK6) is linked with a cyclin partner and plays a crucial role in the early stages of cancer development. It is currently a potential drug target for developing therapeutic molecules targeting cancer therapy. Here, we have identified taurine as an inhibitor of CDK6 using combined in silico and experimental studies. We performed various experiments to find the binding affinity of taurine with CDK6. Molecular docking analysis revealed critical residues of CDK6 that are involved in taurine binding. Fluorescence measurement studies showed that taurine binds to CDK6 with a significant binding affinity, with a binding constant of K = 0.7 × 107 M–1 for the CDK6–taurine complex. Enzyme inhibition assay suggested taurine as a good inhibitor of CDK6 possessing an IC50 value of 4.44 μM. Isothermal titration calorimetry analysis further confirmed a spontaneous binding of taurine with CDK6 and delineated the thermodynamic parameters for the CDK6–taurine system. Altogether, this study established taurine as a CDK6 inhibitor, providing a base for using taurine and its derivatives in CDK6-associated cancer and other diseases.

The antioxidant activities, inhibitory effects, kinetics, and mechanisms of artocarpin and α-mangostin on α-glucosidase and α-amylase

This study investigated the antioxidant activities, enzyme inhibitory activities and the interaction mechanisms of artocarpin and α-mangostin on α-amylase and α-glucosidase. Results showed that artocarpin and α-mangostin had obvious antioxidant activities and inhibitory activities on α-glucosidase and α-amylase. The inhibitions of the two compounds on α-glucosidase were reversible and non-competitive according to the kinetics studies. Fluorescence intensity measurements indicated that the interaction mechanisms between the inhibitors and the two enzymes were static processes. Isothermal titration calorimetry (ITC) analysis showed that the bindings between the inhibitors and the enzymes complex were all spontaneous. The main driving forces between α-mangostin and artocarpin with α-glucosidase might be hydrogen bonds and electrostatic interactions, respectively. While the forces between the two inhibitors and α-amylase might be hydrophobic interactions. Furthermore, molecular docking results showed that artocarpin and α-mangostin could bind to the allosteric site of the two enzymes, except for artocarpin in the active site pocket of α-amylase. All the results indicated that artocarpin and α-mangostin might be promising candidates for hypoglycemic functional products.

Kinetic Analysis of T4 Polynucleotide Kinase via Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) measures the heat absorbed or released from biochemical processes. Although the primary application of ITC has been in binding studies, the reaction enthalpy detected by ITC can be used as a probe to quantify enzyme activity. Kinases are an important class of enzymes that are involved in many types of cellular regulation, but kinetic studies of kinases by ITC have been limited. T4 polynucleotide kinase (T4 PNK) is proposed as a model system to optimize and validate the ITC analysis conditions for a kinase. T4 PNK typically phosphorylates an oligonucleotide, but a single nucleotide can serve as a substrate if a 3′‐phosphate group is present. We used ITC to study the T4 PNK‐catalyzed conversion of 3′‐AMP to adenosine‐3′,5′‐bisphosphate. To initiate the reaction, a single injection of 3′‐AMP substrate was made into the ITC cell containing T4 PNK and ATP at pH 7.6 and 30oC. The shape of the resultant Differential Power versus Time curve is determined by the kinetic properties of the enzyme. Integration of the area under the curve yields reaction enthalpy. The apparent ∆H for the reaction was found to vary depending on the type of buffer used. This result was explained by differential protonation of reactants and products at pH 7.6, and indicated the release of approximately 0.6 protons per mole for the forward reaction. Extrapolation was used to estimate an intrinsic (i.e. buffer‐independent) ∆H of ‐25 kJ/mol. The Michaelis‐Menten constant, KM, was 1.3E‐05 M, and the turnover number, kcat, was 8/sec. The reaction enthalpy and kinetic parameters for T4 PNK phosphorylation of 3′‐AMP were assessed successfully by single injection ITC analysis. This finding supports the potential to analyze more complex kinase systems using ITC.

Monomerization of abscisic acid receptors through CARKs-mediated phosphorylation.

Cytosolic ABA Receptor Kinases (CARKs) play a pivotal role in abscisic acid (ABA)-dependent pathway in response to dehydration, but their regulatory mechanism in ABA signaling remains unexplored. In this study, we showed that CARK4/5 of CARK family physically interacted with ABA receptors (RCARs/PYR1/PYLs), including RCAR3, RCAR11-RCAR14, while CARK2/7/11 only interacted with RCAR11-RCAR14, but not RCAR3. It indicates that the members in CARK family function redundantly and differentially in ABA signaling. RCAR12 can form heterodimer with RCAR3 in vitro and in vivo. Moreover, the members of CARK family can form homodimer or heterodimer in a kinase activity dependent manner. ITC (isothermal titration calorimetry) analysis demonstrated that the phosphorylation of RCAR12 by CARK1 enhanced the ABA binding affinity. The phosphor-mimic RCAR12T105D significantly displayed ABA-induced inhibition of the phosphatase ABI1 (ABA insensitive 1) activity, leading to upregulation of ABA-responsive genes RD29A and RD29B in cark157:RCAR12T105D transgenic plants, which exhibited ABA hypersensitive phenotype. The transcription factor ABI5 (ABA insensitive 5) activates the transcriptions of CARK1 and CARK3 by binding to ABA-response elements (ABREs) of their promoters. Collectively, our data imply that the dimeric CARKs phosphorylate homodimer or heterodimer ABA receptors, leading to monomerization for triggering ABA responses in Arabidopsis.

Structural and functional study of a novel lytic polysaccharide monooxygenase cPMO2 from compost sample in the oxidative degradation of cellulose

Lytic polysaccharide monooxygenases (LPMOs) are the major groups of oxidizing the recalcitrant polysaccharides. Here, a novel AA9 family protein cPMO2 originated from composting was characterized, and it had a better binding capacity to phosphoric acid swollen cellulose (PASC) and Avicel, as well the isothermal titration calorimetry (ITC) analysis showed that it owned a considerable capacity of binding Cu2+ with the Kd value of 1.58 ± 0.78 μM. Interestingly, the kinetics of cPMO2 by using 2, 6-dimethoxyphenol (2, 6-DMP) as substrate indicated that cPMO2 was a novel member of LPMOs family, with the Vmax of 21.30 ± 0.83 U·g−1 at pH 7.5. In addition, cPMO2 owned a broad substrate specificity, which could act on PASC and xylan at the C1 site based on the MALDI-TOF-MS analysis results. Simultaneously, cPMO2 could also act on Avicel directly resulting the decrease of crystallinity by 4% and further to synergistic degradation of corncob and rice straw with glycoside hydrolases. The structure of the functional region of cPMO2 was obtained by using single-crystal X-ray diffraction, and the enzymatic properties and molecular docking analysis results showed that His1 and Tyr175 were the critical active sites of the cPMO2. Altogether, this study will be a supplement for the AA9 LPMOs in cellulose degradation.

Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail

Eukaryotic mRNAs possess a poly(A) tail at their 3′-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)-dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1–poly(A) and PABPC1–Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM)2–RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, Kd = 1 nM). However, the Kd values of isolated RRM2 were 200 and 4 μM in their interactions with poly(A) and Paip2A, respectively; Kd values of 5 and 1 μM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)-binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.

Vitamin D3 microcapsules formed by heteroprotein complexes obtained from amaranth protein isolates and lactoferrin: Formation, characterization, and bread fortification

Vitamin D3 (VD3) is a fat-soluble compound important for human health but is unstable in the presence of environmental factors such as light and hot temperature. The present work aimed to microencapsulate VD3 in complexes formed by Amaranth protein isolates (APIs) and lactoferrin (Lf). The encapsulation was conducted at pH 6.5 and a ratio of 1:3 (API:Lf, w/w) after confirming these complexes affinity with zeta potential and isothermal titration calorimetry analysis. The microcapsules showed a spherical morphology with sizes below 600 nm and high encapsulation efficiency (up to 90%). Fourier transform infrared spectroscopy data confirmed the encapsulation of VD3. During simulated gastrointestinal digestion of the microcapsules, the highest percentage (70%) of VD3 release was observed in the intestinal phase, with a high bioaccessibility (approximately 46%). Compared to unencapsulated VD3, microencapsulation protected VD3 from degradation under stress conditions (ultraviolet radiation and storage at room temperature) by a factor of up to three times. Furthermore, API: Lf heteroprotein complexes also protected (86%) VD3 during a simulation of bread production. Microcapsules added to bread showed release profile kinetics of the first order when exposed to simulated intestinal fluid. These results suggest that VD3 microcapsules formed as API: Lf complexes can be applied successfully for food fortification and baking.

Molecular Basis of Rhodomyrtone Resistance in Staphylococcus aureus.

ABSTRACTRhodomyrtone (Rom) is a plant-derived broad-spectrum antibiotic active against many Gram-positive pathogens. A single point mutation in the regulatory farR gene (farR*) confers resistance to Rom in Staphylococcus aureus (RomR). The mutation in farR* alters the activity of the regulator, FarR*, in such a way that not only its own gene, farR*, but also the divergently transcribed farE gene and genes controlled by the global regulator, agr, are highly upregulated. Here, we show that mainly the upregulation of the fatty acid efflux pump FarE causes the RomR phenotype, as farE deletion in either the parent or the RomR strain (RomR ΔfarE) yielded hypersensitivity to Rom. Comparative lipidome analysis of the supernatant (exolipidomics) and the pellet fraction revealed that the RomR strain excreted about 10 times more phospholipids (PGs) than the parent strain or the ΔfarE mutants. Since the PG content in the supernatant (2,244 ng/optical density [OD]) was more than 100-fold higher than that of fatty acids (FA), we assumed that PG interacts with Rom, thereby abrogating its antimicrobial activity. Indeed, by static and dynamic light scattering (SLS and DLS) and isothermal titration calorimetry (ITC) analyses, we could demonstrate that both PG and Rom were vesicular and reacted with each other in milliseconds to form a 1:1.49 [Rom-PG(32:0), where PG(32:0) is PG with C32:0 lipids] complex. The binding is entropically driven and hence hydrophobic and of low specificity in nature. Our results indicate that the cytoplasmic membrane is the actual target of Rom, which is also in agreement with Rom’s induced rapid collapse of the membrane potential and decreased membrane integrity.

Characterization of a synthetic zinc-chelating peptide from sea cucumber (Stichopus japonicus) and its gastrointestinal digestion and absorption in vitro.

Zinc absorption in intestinal system could be strongly affected by the gastrointestinal digestion and absorption of zinc-chelating peptides serving as zinc carriers. In this study, a novel zinc-chelating sea cucumber synthetic peptide (SCSP) was synthesized to estimate its gastrointestinal digestion and promotive effect of zinc absorption in vitro. Analysis of isothermal titration calorimetry suggested that the binding of SCSP and zinc (N ≈ 1) was exothermic, with relatively weak binding affinity (K=1.0 × 10-3 mol L-1 ). The formation of SCSP-Zn complexes brought morphological changes to the peptides confirmed by scanning electron microscopy (SEM), which also indicated 6.88% of the existence of zinc element. In addition, the SCSP-Zn complexes remained stable under simulated human gastrointestinal digestion. In an in vitro study, the SCSP-Zn complex could successfully transport through the intestinal membrane in the model of everted rat gut sacs (nearly 7.5μM cm-2 ) as well as Caco-2 cells where the zinc transport reached 0.0014 mg mL-1 carried by SCSP. Fluorescence staining experiments revealed free zinc accumulation inside the tissues and cells treated with the SCSP-Zn complex. The chelation SCSP-Zn had the promotion ability of zinc absorption in vitro and ex vivo experiments, which suggested a theoretical basis for the design and production of effective zinc chelating peptides as zinc carriers to improve zinc bioavailability. © 2022 Society of Chemical Industry.

A microcalorimetric and microscopic strategy to assess the interaction between dietary fibers and small molecules

The interaction between small molecules and neutral soluble dietary fiber is one of the proposed mechanisms determining the bioavailability of these components in the small intestine. However, the weak nature of these interactions makes it difficult to find an analytical method sensitive enough to detect them. Here, we probed the molecular interaction between galactomannan, arabinoxylan, and β-glucan with gallic acid, cinnamic acid, acetylsalicylic acid, and acetaminophen, using advanced analytical methods, namely isothermal titration calorimetry (ITC) and in the form of gold-nanoparticles, transmission electron microscopy (TEM). The results obtained from ITC analysis were fully consistent with the results obtained from TEM. In short, the interaction of these fibers and small molecules was mainly entropically driven, hence involving hydrophobic type association and possible conformational changes of the polysaccharide. However, the enthalpy contribution (hydrogen interaction) is also significant, especially regarding interactions with the acetylsalicylic acid molecule.

2-Amino-1,8-naphthyridine Dimer (ANP77), a High-Affinity Binder to the Internal Loops of C/CC and T/CC Sites in Double-Stranded DNA.

Small molecules targeting DNA regions with structural fluctuation are an important class of molecule as chemical probes for studying the role of these structures in biological systems and the development of neurological disorders. The molecule ANP77 we described here, where a three-atom linker connects two 2-amino-1,8-naphthyridines at the C7 position, was found to form stacked structure with protonation of naphthyridine at low pH, and bound to the internal loop consisting of C/CC and T/CC in double-stranded DNA with affinities of 4.8 and 34.4 nM, respectively. Mass spectrometry and isothermal titration calorimetry analyses determined the stoichiometry for the binding as 1:1, and chemical footprinting with permanganate and NMR structural analysis revealed that the T in the T/CC was forced to flip out toward an extrahelical position upon ANP77 binding. Protonated stacked ANP77 interacted with two adjacent cytosines through hydrogen bonding and occupied the position in the duplex by flipping out the C or T opposite CC. Finally, this study demonstrated the potential of ANP77 for binding to the sequences of biological significance with the TG(T/C)CC repeat of the PIG3 promoter and the telomere repeat CCCTAA.

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl β-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

Insights into the impacts of dissolved organic matter of different origins on bioaccumulation and translocation of per- and polyfluoroalkyl substances (PFASs) in wheat

Per- and polyfluoroalkyl substances (PFASs) have been found to be widely present in soil. Dissolved organic matter (DOM) in soil are supposed to greatly affect the bioavailability of PFASs in soil. Herein, hydroponic experiments were conducted to understand the impacts of two kinds of typical DOM, bovine serum albumin (BSA) and humic acid (HA), on the uptake and translocation of legacy PFASs and their emerging alternatives, perfluorooctane sulfonic acid (PFOS), perfluorooctane acid (PFOA), perfluorohexane sulfonic (PFHxS) and 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA) in wheat (Triticum aestivum L.). The results indicated that both HA and BSA significantly inhibited the bioaccumulation and translocation of PFASs in the roots and shoots of wheat, and the impacts of BSA were greater than HA. This difference was explained by the greater binding affinities of the four PFASs with BSA than with HA, as evidenced by the equilibrium dialysis and isothermal titration calorimetry (ITC) analyses. It was noting that inhibition impacts of the BSA-HA mixture (1:1) were lower than BSA alone. The results of Fourier transform infrared (FT-IR) spectroscopy and excitation-emission matrix (EEM) fluorescence spectroscopy suggested that HA could bind with the fluorescent tryptophan residues in BSA greatly, competing the binding sites with PFASs and forming a cover on the surface of BSA. As a result, the binding of PFASs with BSA-HA complex was much lower than that with BSA, but close to HA. The results of this study shed light on the impacts of DOM in soil on the bioaccumulation and translocation of PFASs in plants.

Preparation and characterization of natural extracts-loaded food grade nanoliposomes

The development of food grade liposomes encapsulating citrus extract (CE) and essential oils (EOs) mixture respectively − was carried out by optimising sonication process and liposome composition and by characterizing the interactions between bioactive compounds and liposomes by Fourier-transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Results showed that the use of deoiled lecithin at 1.5% and 10 min-treatment at 70% of full power allowed the formation of more stable nanoliposomes with high EE. Based on ITC analysis, liposome had more affinity to encapsulate CE than EOs. FTIR demonstrated low effects of bioactive loading on the molecular structure of liposomes. The chemical interactions especially involved ethyl, methyl, P–O–C and PO2 - groups. The present system is environmental-friendly with respect to a potential food grade assessment and could be very suitable for food applications.