Tryptophan Fluorescence Emission Research Articles

Development of a high throughput oxidation profiling strategy for monoclonal antibody products

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

Pre-treatment effects on the composition and functionalities of pigeon pea seed ingredients

Soaking and boiling can improve the digestibility of seeds by reducing anti-nutritional factors and the hard-to-cook phenomenon. The use of pre-treated seeds in protein fractionation is thus of interest for the development of protein-rich ingredients. In this work, soaked and soaked-boiled seeds were applied prior to aqueous fractionation of pigeon pea proteins. Pre-treatments impact the particle size distribution in the flours, with boiling leading to aggregate formation (>1000 μm) due to starch gelatinization. The soaked-boiled flour showed a lower lipid content, higher content in soluble polysaccharides, and loss of proteins with low molecular weight compared to the control. The use of soaked-boiled seeds for protein fractionation reduced trypsin inhibitor activity by 95%, demonstrating a positive effect in improving nutritional quality. Protein fractionation also resulted in a 3-fold accumulation of lipids, and the mannose content, notably, doubled in protein concentrates compared to their flour counterparts. A lower intensity of tryptophan fluorescence emission was observed in protein concentrates, which may be related to chemical degradation or interactions with other molecules. The protein concentrate from soaked-boiled seeds exhibited higher emulsion stabilization properties compared to the others protein concentrates. The decrease in foaming properties caused by the soaking can be attributed to the leaching of minor compounds that also act as foaming agents, such as saponins. This work brings new insights into the effects of soaking and boiling-based pre-treatments on the composition of pulse protein ingredients, and in turn, on their techno-functionalities as exemplified with their emulsifying, foaming and gelling properties.

Physico-chemical and structural modifications of caseins in micellar casein isolate induced by pulsed electric field

The potential of pulsed electric field (PEF) to modify the structure of caseins in micellar casein isolate (MCI) was investigated. MCI was PEF-treated at room (RT, 23 °C) and cold (CT, 4 °C) temperatures, using an electric field strength of 16 kV cm−1 for 6 or 31 μs. Conditions simulating elderly digestion, such as the peptide profile released after gastric digestion and bioactivity, were studied. Unfolding of the protein structure was observed in PEF-treated MCI at RT, which led to cleavage points that were more accessible to enzymes. By contrast, fluorescence emission of tyrosine and tryptophan and Raman spectra indicated a reorganization in the configuration of the micelle in PEF-processed at CT. Under these conditions was observed an increase in the number of peptides generated from gastric digestion compared with untreated MCI, especially the bioactive peptides FSDKIAK f(31–45) and MPFPKYPVEP f(124–133), despite a lower overall uptake of bioactive peptides by Caco-2 cells.Industrial relevance: Micellar casein isolate (MCI) is widely utilized for protein fortification (e.g., in fresh dairy products, smoothies, meat products, and clinical nutrition) and for the adjustment of functional properties in food (e.g., viscosity, foaming). The findings of this work provide valuable information about the potential of PEF processing to modify the properties of MCI.

PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life.

Recombinant urate oxidase (UOX, E.C.1.7.3.3) is an important therapeutic enzyme used in preventing and treating chemotherapy-induced hyperuricemia and severe gout. However, UOX use is limited due to the poor stability and short plasma half-life. To solve this problem, we designed three PASylated variants of Aspergillus flavus UOX with different PAS sequences at the C- or N-terminus. The genes of native and PASylated variants (UOX-PAS20, PAS24-UOX, and UOX-PAS100) were designed and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant native and PASylated enzymes were compared in terms of biophysical properties, kinetics parameters, and pharmacokinetics behavior using standard methods. PASylation of UOX with PAS100 polymer caused a 1.24-fold reduction in K m to 52.61 μM, and a 3.87-fold increase in K cat/K m for uric acid compared to the native variant. UOX-PAS100 retained its activity in different temperatures (20-55 °C); however, other variants lost nearly 50% of their original activity at 55 °C. UOX-PAS100 exhibited a 1.78-fold increase in hydrodynamic radius and a 1.64-fold larger apparent molecular size in comparison to the native UOX. Circular dichroism (CD) spectroscopy demonstrated that the addition of the PAS tag does not change the secondary structure of the fusion enzyme. The tryptophan fluorescence emission spectra for PASylated enzymes showed a significant modification in the conformational state of UOX by the PAS polymer presence. UOX-PAS100 retained 89.0% of the original activity following 72 h incubation in the presence of plasma at 37 °C. However, only about 61.0%, 57.0%, 50.0%, and 52.0% of activity from PAS24-UOX, UOX-PAS20, native UOX, and rasburicase (Fasturtec, Italy) remained, respectively, at the identical time. UOX-PAS100 had an increased biological half-life (8.21 h) when compared with the rasburicase (3.12 h) and native UOX (2.87 h) after being injected into a rat. Having considering everything, our results suggest that the UOX-PAS100, an A. flavus UOX fused with a C-terminally 100 amino acid PAS-residue, is a proper candidate with enhanced biological activity and extended plasma half-life for clinical therapy in patients suffering from hyperuricemia.

Using Intrinsic Fluorescence to Measure Protein Stability Upon Thermal and Chemical Denaturation.

Understanding how point mutations affect the performance of protein stability has been the focus of several studies all over the years. Intrinsic fluorescence is commonly used to follow protein unfolding since during denaturation, progressive redshifts on tryptophan fluorescence emission are observed. Since the unfolding process (achieved by chemical or physical denaturants) can be considered as two-state N➔D, it is possible to utilize the midpoint unfolding curves (fU=50%) as a parameter to evaluate if the mutation destabilizes wild-type protein. The idea is to determine the [D]1/2 or Tm values from both wild type and mutant and calculate the difference between them. Positive values indicate the mutant is less stable than wild type.

A novel concept of Pyridoxal 5'-phosphate permeability in E.coli for modulating the heterologous expression of PLP dependent proteins

PLP (Pyridoxal 5′-phosphate) dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetylserine sulphdrylase (OASS) of H.contortus and determined that the lysine-47 is very important for the binding of PLP. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli. Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01 mM (Group II), 0.025 mM (Group III), 0.05 mM (Group IV) and 0.1 mM (Group V)]. There was approximately 4.2 %, 7.2 %, 10.5 % and 18 % increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I. We could find a PLP concentration-dependent increase in expression and catalytic activity signifying the probable transport of PLP across the membrane. E.coli does not secrete any dephosphorylation factor for PLP in the immediate microenvironment. There was a significant reduction in levels of inorganic phosphate after PLP dephosphorylation in induced group in comparison to uninduced E.coli and BL21 groups, signifying PLP internalization in E.coli. The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies.

Contribution of Nascent Polypeptides of increasing Length to the Apparent Stability of the Bacterial Ribosome

The role of the ribosome in the cell extends well beyond catalysis of peptide-bond formation. For instance, the ribosome is emerging as a key player in cotranslational protein folding. On the other hand, whether and how the ribosome assists nascent-protein folding, and how the nascent protein may in turn affect the ribosome, is poorly understood. Recent results suggest that the ribosome facilitatesprotein folding by directly interacting with nascent chains. Yet, whether interactions between the ribosome and the nascent chain have any influence on the ribosome, and whether these interactions promote proper protein folding is currently unknown. Via sucrose-gradient assays, we found that empty ribosomes are more sensitive to urea denaturation than ribosomal complexes carrying either aminoacyl-tRNA or nascent-chains. Further, by taking advantage of tryptophan fluorescence emission, we probed the effect of nascent chains of widely variable sequence and length on the apparent stability of ribosomal proteins (rProteins). We found that there is no statistically significant difference between the contribution of different nascent chains, suggesting that nascent-chain-to-rProtein interactions are either weak or thermodynamically compensated. Hence, there is no difference between the sensitivity to urea of ribosomal initiation complexes carrying aminoacyl tRNA and ribosomal complexes harboring peptidyl tRNA. in all, the above findings lead us to conclude that aminoacyl tRNA, likely in conjunction with mRNA, increases the apparent stability of the ribosome, while the nascent polypeptide does not. Finally, our data led us to propose a multi-step model for the disassembly of ribosome-nascent-chain complexes.

Characterization of Bio-Inspired Electro-Conductive Soy Protein Films.

Protein-based conductive materials are gaining attention as alternative components of electronic devices for value-added applications. In this regard, soy protein isolate (SPI) was processed by extrusion in order to obtain SPI pellets, subsequently molded into SPI films by hot pressing, resulting in homogeneous and transparent films, as shown by scanning electron microscopy and UV-vis spectroscopy analyses, respectively. During processing, SPI denatured and refolded through intermolecular interactions with glycerol, causing a major exposition of tryptophan residues and fluorescence emission, affecting charge distribution and electron transport properties. Regarding electrical conductivity, the value found (9.889 × 10−4 S/m) is characteristic of electrical semiconductors, such as silicon, and higher than that found for other natural polymers. Additionally, the behavior of the films in contact with water was analyzed, indicating a controlled swelling and a hydrolytic surface, which is of great relevance for cell adhesion and spreading. In fact, cytotoxicity studies showed that the developed SPI films were biocompatible, according to the guidelines for the biological evaluation of medical devices. Therefore, these SPI films are uniquely suited as bioelectronics because they conduct both ionic and electronic currents, which is not accessible for the traditional metallic conductors.

Assembly and Characterization of Biocompatible Coenzyme Q10 -Enriched Lipid Nanoparticles.

Coenzyme Q10 (CoQ10 ) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ10 was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ10 nanodisks (ND), contain 1.0 mg CoQ10 /5 mg PtdCho/2 mg apoA-I (97% CoQ10 solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ10 ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ10 into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ10 ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ10 . Incubation of cultured cells with CoQ10 ND, but not empty ND, resulted in a significant increase in the CoQ10 content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ10 in lipid nanoparticles has been developed. The availability of CoQ10 ND provides a novel means to investigate biochemical aspects of CoQ10 uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.

Effect of Nascent Proteins on the Stability of the Ribosome

Recent studies showed that many nascent proteins begin folding during translation, sometimes within the vestibule of the ribosomal exit tunnel. On the other hand, how and whether the ribosome assists protein folding, and how protein folding may in turn affect the ribosome, is poorly understood. We recently detected interactions between foldable and intrinsically disordered nascent proteins (not bearing an N-terminal signal sequence) and ribosomal proteins by dynamic fluorescence depolarization in the frequency domain and by chemical cross-linking. These results suggest that the ribosome facilitates in vivo protein folding by directly interacting with nascent chains. Whether interactions between the ribosome and the nascent chain have any thermodynamic influence on the ribosome, and whether these interactions promote proper protein folding however, is not known. Via sucrose-gradient assays, we found that empty ribosomes are less stable than ribosomes carrying tRNA, or ribosomes harboring nascent-chains. In addition, by taking advantage of tryptophan fluorescence emission, we probed the effect of several nascent chains of variable sequence and length on the stability of ribosomal proteins. We found that there is no statistically significant difference between the contribution of different nascent-chain constructs, suggesting that nascent-to-ribosomal protein interactions are either weak or thermodynamically compensated. Hence, there is no apparent difference between the overall stability of ribosomes carrying a tRNA and ribosomes harboring a nascent-chain. In all, the above findings lead us to conclude that tRNA stabilizes the ribosome, while nascent proteins do not. Therefore, intriguingly, the interplay between the ribosome and nascent chains does not critically rely on standard-state net free-energy changes. Finally, our data led us to propose a multi-step model for the disassembly of ribosome-nascent-chain complexes.

Bluetongue virus viral protein 7 stability in the presence of glycerol and sodium chloride

PurposeThe Orbivirus Bluetongue virus (BTV) is an economically significant disease that affects mainly wild and domestic ruminants. BTV is most often seen symptomatically in sheep, but is easily carried by goats, cattle, and wild ruminants. To date there are several problems with the vaccines currently available for BTV, and one of the most promising candidates to increase vaccine efficacy is a protein-based vaccine, for which viral protein 7 (VP7) is a great candidate to be included in it. In order to further these studies, the stability of BTV VP7 in common vaccine additives needs to be investigated.Materials and MethodsRecombinant BTV VP7 was expressed in a bacterial cell system and purified before being analysed using spectroscopic techniques including far-ultraviolet (UV) circular dichroism and intrinsic tryptophan fluorescence. BTV was analysed in a number of different buffer conditions.ResultsWe report here that BTV VP7 maintains its native secondary structure until at least 52℃ and native-like tertiary structure to at least 80℃. Far-UV circular dichroism and intrinsic tryptophan fluorescence emission spectra indicate significant secondary and tertiary structure remaining even at 90℃, respectively. Six M guanidinium chloride is able to unfold BTV VP7 while 8 M urea could not.ConclusionTwenty percent glycerol and 300 mM sodium chloride appear to have a protective effect on BTV VP7's structure, as significantly more structure is seen at 90℃ when compared to BTV VP7 without the addition of these chemicals. Both glycerol and sodium chloride are common vaccine additives.

Quenching of tryptophan fluorescence by colloidal Cu2S nanoparticles through static and dynamic modes under different solution pH

The study of the interaction between biomolecules and nanoparticles (NPs) is interesting since the dimensions of the two overlap to some extent. Herein, the interaction of an essential amino acid, tryptophan, with Cu2S NPs was investigated by using various spectroscopic techniques. The 220 nm absorption band of tryptophan was found to be blue-shifted by 2 and 5 nm at pH 6 and 10.6 respectively. The binding of tryptophan with the Cu2S NPs at pH 10.6 via its indole chromophore was established by the Fourier transform infrared (FTIR) spectroscopic measurements. The fluorescence emission of tryptophan was quenched by the semiconductor NPs. The fluorescence quenching was found to be dynamic in nature at pH 6. However, the static mode of quenching was found to be operative at pH 10.6. The binding constants and thermodynamic parameters were calculated for the tryptophan-Cu2S NPs complex at pH 10.6.

Polynuclear zinc(II) complexes of thiosemicarbazone: Synthesis, X-ray structure and biological evaluation

Two new dimeric Zn(II) ([{ZnL1(DMSO2)}2]·DMSO (1), [{ZnL2Cl}2] (2)) and a novel tetrameric Zn(II) complex ([(Zn2L3)2(μ–OAc)2(μ3–O)2] (3)), where H2L1 = 4–(p–methoxyphenyl) thiosemicarbazone of o–hydroxynapthaldehyde, HL2 = 4–(p–methoxyphenyl)thiosemicarbazone of benzoyl pyridine and H2L3 = 4–(p–chlorophenyl)thiosemicarbazone of o–vanillin are reported. Ligands and their complexes were characterized by spectroscopic and single crystal X–ray diffraction techniques. In addition, the complexes exhibited good binding affinity towards HSA (1012 M−1), which is supported by their ability to quench the tryptophan fluorescence emission spectra of HSA. The complexes were also screened for their DNA binding propensity through UV–vis absorption titration, circular dichroism and fluorescence spectral studies. Results show that they effectively interact with CT–DNA through an intercalative mode of binding, with binding constants ranging from 103 to 104 M−1. Among the three complexes 1 has the highest binding affinity towards CT–DNA. Further, the phosphatase activity was evaluated using bis(2,4–dinitrophenyl)phosphate (BDNPP) as substrate, however, the complexes did not yield any measurable catalytic activity. Nevertheless the complexes showed significant cytotoxic potential against HeLa and HT–29 cancer cell lines that was assessed through MTT assay and DAPI staining. Remarkably, complex 1 showed better activity than cisplatin against HT–29 cell line.

Interaction of a Model Hydrophobic Drug Dimethylcurcumin with Albumin Nanoparticles.

The aim of present study was to investigate the binding interactions of a model hydrophobic molecule, dimethylcurcumin (DMC) with nanoparticle form of bovine serum albumin (BSA) using fluorescence spectroscopy techniques. For this, BSA nanoparticles (size = 62.0 ± 3.5nm, molecular weight = 11,243 ± 3445 kD) prepared by thermal denaturation method was mixed with DMC in solution and monitored for fluorescence emission of tryptophan (Trp) residue as well as DMC separately. The emission maximum of DMC in nanoparticles form exhibited more blue sift and quenched the excited state of tryptophan (Trp) by six fold higher than in the native form of BSA. By analyzing Trp fluorescence, the mean binding constant (K) estimated for the interaction of DMC with native and nanoparticles forms of BSA was 2.7 ± 0.4 × 104M-1 and 1.5 ± 0.5 × 105M-1 respectively. Together these results suggested that DMC experienced a more rigid environment in nanoparticles than in native form of BSA. Additionally the above determined K values were in agreement with those reported previously by absorption techniques. Further direct energy transfer was observed between Trp and DMC, using which the distance (r) calculated between them was 28.25 ± 0.27Ǻ in BSA native. Similar analysis involving BSA nanoparticle and DMC revealed a distance of 24.25 ± 1.05Ǻ between the hydrophobic core and the ligand. Finally interaction of DMC with BSA was validated through molecular docking studies, which indicated sub-domain IIA as the binding site of DMC. Thus it is concluded that intrinsic fluorescence of protein can be utilized to study the interaction of its different physical forms with any hydrophobic ligand.

Structural insights into the binding behavior of isoflavonoid glabridin with human serum albumin

Glabridin (GB), a prenylated isoflavonoid in licorice root has been used as foods and medicines with beneficial biological activities. Human serum albumin (HSA) is the most abundant blood plasma protein binding and transporting ligands such as GB. The present study investigated the interaction between GB and HSA using multi-spectroscopic and molecular docking techniques. Fluorescence spectroscopy demonstrated that GB strongly quenched the tryptophan fluorescence emission of HSA, suggesting that GB-HSA interaction followed a static mode of quenching with a binding constant (K, 105-104 M−1, 25-37 °C) reflecting strong-to-moderate affinity of GB to HSA. Molecular displacement and protein-ligand docking simulations showed that the probable GB-binding positions of HSA exist near the site I of HSA. The hydrophobic interaction is a dominant binding force for forming GB-HSA complexes, and the binding resulted in conformational changes of HSA protein with more β-sheet structures. Furthermore, the complex formation of GB with HSA changed the hydrodynamic size and zeta-potential of HSA with enhanced solubility of GB. The elucidation of GB-HSA binding behavior will provide insights into the application of GB in food and pharmaceutical industries.

High-level expression and molecular characterization of a recombinant prolidase from Escherichia coli NovaBlue.

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene from Escherichia coli NovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-β-D-galactoside IPTG-induced E. coli M15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass of EcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris–HCl buffer (pH 8.0) as a dimeric form. The optimal conditions for EcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ ion. Kinetic analysis with Ala-Pro as the substrate showed that the Km and kcat values of EcPepQ were 8.8 mM and 926.5 ± 2.0 s−1, respectively. The thermal unfolding of EcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-U value of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity of EcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability of EcPepQ allows to adjust the reaction conditions more suitable for EcPepQ-catalyzed bioprocess.

The Stability, Reduction Potential and Ligand State of Two Conformations of a C-Type Cytochrome from the Diatom Thalassiosira Pseudonana

In this study we investigate the structure and function of a c-type cytochrome from the diatom Thalassiosira pseudonana, Tp34211. The axial iron ligand of a c-type cytochrome is often either a methionine or histidine residue. The axial ligand plays an important role in determining the midpoint potential of the heme cofactor, which in turn determines the possible physiological electron donors and acceptors. Initial experiments suggested that recombinantly expressed Tp34211 forms two alternative conformations that can be differentiated by Soret band changes upon imidazole binding or quenching of the tryptophan fluorescence emission signal. Based on sequence alignments of Tp34211 and known cytochrome c6 proteins, we predict that Tp34211 has an axial histidine ligand (H78), but a neighboring lysine residue (K79) could serve as an alternate ligand. To test the possible role of ligand switching in the two alternative conformations observed for Tp34211, we recombinantly expressed and purified both wild-type Tp34211 and a K79A variant that should force bis-His ligation. Following purification by ion-exchange and size-exclusion chromatogrpahy, guanidine induced unfolding assays were performed on the various protein samples, with extent of unfolding measured via intrinsic tryptophan fluorescence. The ΔG°unfolding of the unquenched conformation of WT Tp34211 and the K79A mutant were determined to be the same within error, suggesting that histidine coordination is favored under normal solution conditions. Midpoint potentials of the K79A variant and the two conformations of the wild-type protein will also be measured to further identify the role of histidine versus lysine ligation in Tp34211.

Effect of curcumin on amyloid-like aggregates generated from methionine-oxidized apolipoprotein A-I.

Curcumin is a polyphenolic phytonutrient that has antineurodegenerative properties. In this study, we investigated the anti‐amyloidogenic properties of curcumin. Following incubation with curcumin, intrinsic tryptophan fluorescence emission of apolipoprotein (apo) A‐I was strongly quenched. At the same time, curcumin fluorescence emission was enhanced. The fluorescence emission spectra of curcumin in the presence of amyloid‐like aggregates formed by methionine‐oxidized (ox) apoA‐I varied, depending on whether curcumin was added before, or after, aggregate formation. The impact of curcumin on the structure of the aggregating material was revealed by the lower amount of β‐structure in ox‐apoA‐I amyloid‐like aggregates formed in the presence of curcumin, compared to aggregates formed without curcumin. However, the kinetics of ox‐apoA‐I amyloid‐like aggregate formation was not altered by the presence of curcumin. Moreover, electron microscopy analysis detected no discernable differences in amyloid morphology when ox‐apoA‐I amyloid‐like aggregates were formed in the presence or absence of curcumin. In conclusion, curcumin interacts with apoA‐I and alters the structure of ox‐apoA‐I amyloid‐like aggregates yet does not diminish the propensity of ox‐apoA‐I to form aggregates.

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Synthesis, characterization and separation of chiral and achiral diastereomers of Schiff base Pd(II) complex: A comparative study of their DNA- and HSA-binding

A racemic mixture of a new chiral Schiff base ligand (HL: R/S-(1-phenylethylimino)methylnaphtalen-2-ol) has been utilized to prepare Pd(II) complex. Crystallization technique has been employed to separate diastereomeric pairs of Pd(II) complex: (mesoPdL2) and (racPdL2) that in this paper are known as PdL21 and PdL22, respectively. The synthesized complexes have been characterized by means of elemental analysis (CHN), FT-IR, 1H and 13C NMR spectroscopies. Moreover, PdL21 has been structurally characterized by single-crystal X-ray diffraction. The geometry around the metal center is square-planar. The interaction of two diastereomers of Pd(II) complex with FS-DNA has been explored, using UV–vis spectroscopy, fluorescence quenching, chemometrics and viscosity measurement methods. The PdL21 exhibited higher binding constant, about 10-fold, (1.0×106M−1) as compared to PdL22 (1.51.5×105M−1). Moreover, the human serum albumin (HSA) binding ability has been monitored by absorption, quenching of tryptophan fluorescence emission and circular dichroism (CD) studies. The slight difference is observed between HSA binding affinity with the complexes: PdL21 (6.2×104M−1) and PdL22 (3.3×104M−1). Also, the thermodynamic parameters were determined at three different temperatures (298, 308 and 318K).In this study, molecular docking was also carried out to confirm and illustrate the specific DNA- and HSA-binding of the Pd(II) complexes. In the PdL21-HSA system a T-shaped π-π interaction with PHE206 was observed. While in the PdL22-HSA system there are a hydrogen bond, a π-cation and two T-shaped π-π interactions with ASB324, LYS212 and PHE228, respectively. The groove binding mode of DNA interaction has been proposed for both diastereomers.