Electron Circular Dichroism Research Articles

Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion

AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.

Dihydro-β-agarofuran sesquiterpenoid derivatives with anti-inflammatory activity from the leaves of Tripterygium wilfordii

Triptersinoids A-E (1–5), five undescribed highly oxygenated dihydro-β-agarofuran type sesquiterpenoid polyesters, along with three known analogues were isolated from the aqueous EtOH extracts of the dried leaves of Tripterygium wilfordii. Spectroscopic techniques were used for the elucidation of their chemical structures, and the absolute configurations determined by means of electron circular dichroism (ECD) studies, including octant rule of saturated cyclohexanone and comparison between the experimental and calculated ECD data. These compounds were evaluated for the anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages, and 1α,2α,8β,15-tetraacetoxy-9α-benzoyloxyl-15-nicotinoyloxy-β-dihydroagarofuran (7) and angulateoid B (8) showed potential inhibitory effects with IC50 values of 17.30 ± 1.07 μM and 20.79 ± 1.55 μM.

Penicamide A, A Unique N,N'-Ketal Quinazolinone Alkaloid from Ascidian-Derived Fungus Penicillium sp. 4829.

Previously unreported N,N′-ketal quinazolinone enantiomers [(−)-1 and (+)-1] and a new biogenetically related compound (2), along with six known compounds, 2-pyrovoylaminobenzamide (3), N-(2-hydroxypropanoyl)-2 amino benzoic acid amide (4), pseurotin A (5), niacinamide (6), citreohybridonol (7), citreohybridone C (8) were isolated from the ascidian-derived fungus Penicillium sp. 4829 in wheat solid-substrate medium culture. Their structures were elucidated by a combination of spectroscopic analyses (1D and 2D NMR and Electron Circular Dichroism data) and X-ray crystallography. The enantiomeric pair of 1 is the first example of naturally occurring N,N′-ketal quinazolinone possessing a unique tetracyclic system having 4-quinazolinone fused with tetrahydroisoquinoline moiety. The enantiomeric mixtures of 1 displayed an inhibitory effect on NO production in lipopolysaccharide-activated RAW264.7 cells, while the optically pure (–)-1 showed better inhibitory effect than (+)-1.

Probing Metal Ion Discrimination in a Protein Designed to Bind Uranyl Cation With Femtomolar Affinity.

The design of metal-binding sites in proteins that combine high affinity with high selectivity for the desired metal ion remains a challenging goal. Recently, a protein designed to display femtomolar affinity for , dubbed “Super Uranyl-binding Protein” (SUP), was described, with potential applications for removing in water. Although it discriminated most metal ions present in seawater, the protein showed a surprisingly high affinity for Cu2+ ions. Here, we have investigated Cu2+ binding to SUP using a combination of electron paramagnetic resonance, fluorescence and circular dichroism spectroscopies. Our results provide evidence for two Cu2+ binding sites on SUP that are distinct from the binding site, but one of which interferes with binding. They further suggest that in solution the protein's secondary structure changes significantly in response to binding ; in contrast, the crystal structures of the apo- and holo-protein are almost superimposable. These results provide insights for further improving the selectivity of SUP for , paving the way toward protein-based biomaterials for decontamination and/or recovery of uranium.

RNA modulates aggregation of the recombinant mammalian prion protein by direct interaction

Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general.

Anti-Inflammatory Cembrane-Type Diterpenoids and Prostaglandins from Soft Coral Lobophytum sarcophytoides.

Two new cembrane-type diterpenoids, lobophytins A (1) and B (3), and four new prostaglandins, (5E)-PGB2 (10), (5E)-13,14-dihydro-PGB2 (11), 13,14-dihydro-PGB2 (12) and 13,14-dihydro-PGB2-Me (13), together with ten known compounds were isolated from the soft coral Lobophytum sarcophytoides. The structures of these new secondary metabolites were identified by high resolution mass spectrometry (HR-ESIMS), nuclear magnetic resonance (NMR) and electron circular dichroism (ECD) analyses, as well as the modified Mosher’s method. Compounds 6, 7, 9, 10, 12, 13, 15 and 16 showed potential anti-inflammatory activity by inhibiting the production of nitric oxide (NO) in RAW264.7 cells that were activated by lipopolysaccharide, with IC50 values ranging from 7.1 to 32.1 μM and were better than the positive control indomethacin, IC50 = 39.8 μM.

Design and Fabrication of a Silver Nanocluster-Based Aptasensor for Lysozyme Detection

Lysozyme is a universal part of the innate immune system and is present in various bodily fluids. It is also vastly studied as a biomarker for a variety of diseases and is used in the food industry as a preservative. However, due to lack of a universal and precise detection method, new techniques are highly sought after for this purpose. Here, we introduce a silver nanocluster-based aptasensor to detect lysozyme with high sensitivity. The sensor consists of an RNA aptamer and a complementary DNA sequence with a silver nanocluster template. Silver nanoclusters were synthesized by reduction of silver nitrate in the presence of complementary DNA sequence and characterized by transmission electron microscopy, UV-Visible, fluorescence, and circular dichroism spectroscopy. Emission of the silver nanoclusters switched between low and high intensity based on the quenching ability of the aptamer for DNA-silver nanoclusters and the binding affinity of the aptamer for lysozyme. In the presence of the aptamer, DNA-silver nanoclusters show low fluorescence due to the hybridization of the aptamer, and hence disruption in the formation of DNA-silver nanoclusters. Upon addition of lysozyme, aptamer binding to the DNA-templated silver nanoclusters will be inhibited due to the stronger interaction between the aptamer and lysozyme. Presence of the free DNA template leads to a restored fluorescence signal of DNA-silver nanoclusters which is dependent upon the lysozyme concentration. By utilizing this strategy, the complementary DNA-templated silver nanoclusters sensor can detect concentrations of lysozyme lower than 10 nM via a signal in the visible spectral region. The proposed sensor presents valuable features such as low-cost, high sensitivity, easy fabrication, and convenient operation for detection of lysozyme.

Synthesis and self-assembly of chiral Gemini-shaped hexabenzocoronene amphiphiles

Herein, we focus on constructing Gemini-shaped amphiphilic hexa-peri-hexabenzocoronene (HBC) derivatives, CHBCs and FHBCs, carrying chiral hydrophilic substituents. Compared with CHBCs, FHBCs bears four fluorine substituents at the periphery of the core. The self-assembly behaviors of the HBCs were systematically investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and circular dichroism (CD) spectra. CHBCs self-assembling to form a nanotubular structure under certain conditions, while FHBCs self-assembling to give non-tubular aggregates. All the ordered assemblies formed by CHBCs and FHBCs are CD-active.

Tau Interacts with the C-Terminal Region of α-Synuclein, Promoting Formation of Toxic Aggregates with Distinct Molecular Conformations.

An increasing body of evidence suggests that aggregation-prone proteins associated with various neurodegenerative diseases synergistically promote their mutual aggregation, leading to the co-occurrence of multiple neurodegenerative diseases in the same patient. Here we investigated teh molecular basis of synergistic interactions between the two pathological proteins, tau and α-synuclein, using various biophysical techniques including transmission electron microscopy (TEM), circular dichroism (CD), and solution and solid-state NMR. Our biophysical analyses of α-synuclein aggregation in the absence and presence of tau reveal that tau monomers promote the formation of α-synuclein oligomers and subsequently fibril formation. Solution NMR results also indicate that monomeric forms of tau selectively interact with the C-terminal region of the α-synuclein monomer, accelerating α-synuclein aggregation. In addition, a combined use of TEM and solid-state NMR spectroscopy reveals that the synergistic interactions lead to the formation of toxic α-synuclein aggregates with a distinct morphology and molecular conformation. The filamentous α-synuclein aggregates as well as α-synuclein monomers were also able to induce tau aggregation.

Sesquiterpenes from an Endophytic Aspergillus flavus.

Eight new cadinene-sesquiterpenes (1-8), one eudesmane-sesquiterpene (9), and three known compounds (10-13) were isolated from an endophytic fungus, Aspergillus flavus, which was isolated from a toxic medicinal plant, Tylophora ovata. Their structures were elucidated by interpretation of spectroscopic data, and absolute configurations determined according to the specific rotation and electron circular dichroism methods. Compounds 4-8, 11, and 12 exhibited latent hepatic protection effects at 10 μM, and compound 12 selectively inhibited the proliferation of MCF-7 breast cancer cells with an IC50 values of 2.6 μM.

Fusion proteins consisting of Bet v 1 and Phl p 5 form IgE-reactive aggregates with reduced allergenic activity

The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.

Supramolecular Tripeptide Hydrogel Assembly with 5-Fluorouracil.

In this work, we present Thioflavin T fluorescence, transmission electron microscopy (TEM), circular dichroism (CD), Fourier-transformed infrared (FT-IR), and oscillatory rheometry studies applied to an antineoplastic drug, 5-fluorouracil (5-FU), embedded in a heterochiral tripeptide hydrogel to obtain a drug delivery supramolecular system. The release of 5-fluorouracil was monitored over time by reverse-phase high-performance liquid chromatography (HPLC) and its interaction with the tripeptide assemblies was probed by all-atom molecular dynamics simulations.

Resolution-of-identity accelerated relativistic two- and four-component electron dynamics approach to chiroptical spectroscopies.

We present an implementation and application of electron dynamics based on real-time time-dependent density functional theory (RT-TDDFT) and relativistic 2-component X2C and 4-component Dirac-Coulomb (4c) Hamiltonians to the calculation of electron circular dichroism and optical rotatory dispersion spectra. In addition, the resolution-of-identity approximation for the Coulomb term (RI-J) is introduced into RT-TDDFT and formulated entirely in terms of complex quaternion algebra. The proposed methodology was assessed on the dimethylchalcogenirane series, C4H8X (X = O, S, Se, Te, Po, Lv), and the spectra obtained by non-relativistic and relativistic methods start to disagree for Se and Te, while dramatic differences are observed for Po and Lv. The X2C approach, even in its simplest one-particle form, reproduces the reference 4c results surprisingly well across the entire series while offering an 8-fold speed-up of the simulations. An overall acceleration of RT-TDDFT by means of X2C and RI-J increases with system size and approaches a factor of almost 25 when compared to the full 4c treatment, without compromising the accuracy of the final spectra. These results suggest that one-particle X2C electron dynamics with RI-J acceleration is an attractive method for the calculation of chiroptical spectra in the valence region.

C-Terminal Residue of Ultrashort Peptides Impacts on Molecular Self-Assembly, Hydrogelation, and Interaction with Small-Molecule Drugs

Single molecular changes on a tripeptide can have dramatic effects on their self-assembly and hydrogelation. Herein, we explore C-terminal residue variation on two consistent ultrashort peptide backbones, i.e. acetylated-Leu-Ile-Val-Ala-Gly-Xaa and acetylated-Ile-Val-Xaa (Xaa = His, Arg, Asn). The objective of this study is to identify candidates that can form hydrogels for small-molecule drug (SMD) delivery. Haemolysis and cytotoxicity (with human adipose-derived mesenchymal stem cells) assays showed that the new soluble peptides (Xaa = His, Arg) are cytocompatible. Gelation studies showed that all but acetylated-Ile-Val-Arg could gel under physiological conditions. Longer peptidic backbones drive self-assembly more effectively as reflected in field emission scanning electron microscopy (FESEM) and circular dichroism spectroscopy studies. Rheological studies revealed that the resultant hydrogels have varying stiffness and yield stress, depending on the backbone and C-terminal residue. Visible spectroscopy-based elution studies with SMDs (naltrexone, methotrexate, doxorubicin) showed that besides the C-terminal residue, the shape of the SMD also determines the rate and extent of SMD elution. Based on the elution assays, infrared spectroscopy, and FESEM, we propose models for the peptide fibril-SMD interaction. Our findings highlight the importance of matching the molecular properties of the self-assembling peptide and SMD in order to achieve the desired SMD release profile.

Chemical constituents from Daphne tangutica and their cytotoxicity against nasopharyngeal carcinoma cells

Two new sesquiterpenoids (1−2), together with 30 known compounds including one sesquiterpenoid (3), six diterpenoids (4−9), fourteen lignans (10−23), and nine other kinds of compounds (24−32), were isolated from the stems of Daphne tangutica Maxim. Their structures were determined through extensive spectroscopic analyses, and the absolute configuration of daphnoid A (1) and B (2) were determined by the experimental and calculated electron circular dichroism (ECD) spectra. All the isolates were evaluated against two human nasopharyngeal carcinoma cells (HONE-1 and SUNE-1). Compound 25 (daphnenone) showed potent cytotoxicity toward HONE-1 and SUNE-1with IC50 values of 2.23 and 1.43 μM, respectively. Further studies indicated that compound 25 exhibited cytotoxic effects by inducing tumor cell apoptosis and arresting the cell cycle at G2/M phases in HONE-1 cells.

Antibacterial properties of human beta defensin-3 derivative: CHRG01

Antibiotic resistance in bacteria is a major health concern. Antimicrobial peptides (AMPs) are a class of peptides that are efficient in killing most microbes yet development of resistance to AMPs is rare. However, complex secondary and tertiary structures and difficulties in isolating AMPs have limited their use as antibiotics. It has been demonstrated earlier that small peptides derived from human β defensin-3 (HBD-3) also show antibacterial activity. Here, we perform a detailed characterization of the antibacterial activity of one such derivative: CHRG01. While HBD-3 has 45 amino acids with three disulphide bonds and a β-sheet folded structure, CHRG01 has only 14 amino acids with the cysteine residues replaced by serine. The antibacterial nature of CHRG01 was studied using scanning electron microscopy (SEM), confocal microscopy, circular dichroism (CD) and small-angle X-ray scattering (SAXS). CD data show that CHRG01 is random coiled in solution. SEM and confocal studies show that the mode of action of CHRG01 is pore forming. SAXS studies show that CHRG01 induces a negative Gaussian curvature, the type of curvature needed for pore formation. The above results show that CHRG01, a small peptide without any complex structure, is capable of killing bacteria by permeabilizing their outer membranes.

Structurally Diverse Polyketides From the Mangrove-Derived Fungus Diaporthe sp. SCSIO 41011 With Their Anti-influenza A Virus Activities.

Influenza A virus (IAV) is a severe worldwide threat to public health and economic development due to its high morbidity and mortality. Marine-derived fungi have been evidenced as a prolific source for the discovery of pharmacologically-active lead compounds. During the course of our search for novel bioactive substances from marine microorganisms, six new polyketides, including two octaketides (1–2), one chromone derivative (13), two highly substituted phthalides (17–18), and one α-pyrone derivative (21) along with 22 known congeners were isolated from a mangrove-associated fungus Diaporthe sp. SCSIO 41011. Their structures were determined by spectroscopic analysis and by comparison with literature data. And the absolute configurations were established according to the specific rotation or electron circular dichroism method. Antiviral evaluation results revealed that compounds 14, 15, 26, and 5-chloroisorotiorin displayed significant anti-IAV activities against three influenza A virus subtypes, including A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2), with IC50 values in the range of 2.52–39.97 μM. The preliminary structure-activity relationships (SARs) are also discussed. These findings expand the chemical and bioactive diversity of polyketides derived from the genus Diaporthe, and also provide a basis for further development and utilization of chromone, xanthone, and chloroazaphilone derivatives as source of potential anti-viral chemotherapy agents.

Spectroscopic and molecular modelling studies on glycation modified bovine serum albumin with cyanidin-3-O-glucoside

In this study, we report the glycation mediated effect of bovine serum albumin (BSA) on the molecular interaction mechanism of cyanidin-3-O-glucoside (C3G) by molecular modelling, Uv–visible spectroscopy, transmission electron microscopy (TEM), fluorescence spectroscopy, and circular dichroism spectroscopy studies. The structures of advanced glycation end-products (AGEs) modified BSA were modelled, energy minimized and analyzed for binding affinity by molecular docking studies using Autodock Vina. Glycation experiments are carried out using glucose and methylglyoxal to validate the molecular modelling results on the interaction of modified BSA with C3G. The modified structures were characterized by reduction in the binding pocket volume, surface, depth, hydrophobicity, and hydrogen bond donors/acceptors. Arg-194, Arg-196, Arg-198, Arg-217, Arg-409, Lys-114, Lys-116, Lys-204, Lys 221, and Lys-439 were found to be crucial in the context of glycation of BSA. TEM images represented the formation of unique globular aggregates in the event of glycation. Uv–visible spectroscopic studies showed the formation of new chromophores between 300 and 400 nm in the event of glycation. Fluorescence quenching was observed in a differential manner in the presence of C3G on glycation modified BSA. Circular dichroism studies suggested the loss of helical structure and formation of β-sheeted structure upon glycation, but subsequent C3G binding has resulted in the increase towards helical structure. Our findings suggested that drug binding affinity has been certainly impaired due to glycation and subsequent AGE modification. Arg-p modification has more austere impact on the structure and would affect the binding properties. We conclude that C3G had differential modulation of binding properties on glycated BSA which can help to protect the stability and bioavailability that has been impaired due to glycation mediated structural changes.

Disruption of the E. coli LptC dimerization interface and characterization of lipopolysaccharide and LptA binding to monomeric LptC.

Lipopolysaccharide (LPS) is an essential element of nearly all Gram-negative bacterial outer membranes and serves to protect the cell from adverse environmental stresses. Seven members of the lipopolysaccharide transport (Lpt) protein family function together to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of bacteria such as Escherichia coli. Each of these proteins has a solved crystal structure, including LptC, which is a largely periplasmic protein that is associated with the IM LptB2 FG complex and anchored to the membrane by an N-terminal helix. LptC directly binds LPS and is hypothesized to be involved in the transfer of LPS to another periplasmic protein, LptA. Purified and in solution, LptC forms a dimer. Here, point mutations designed to disrupt formation of the dimer are characterized using site-directed spin labeling double electron electron resonance (DEER) spectroscopy, light scattering, circular dichroism, and computational modeling. The computational studies reveal the molecular interactions that drive dimerization of LptC and elucidate how the disruptive mutations change this interaction, while the DEER and light scattering studies identify which mutants disrupt the dimer. And, using electron paramagnetic resonance spectroscopy and comparing the results to the previous quantitative characterization of the interactions between dimeric LptC and LPS and LptA, the functional consequences of monomeric LptC were also determined. These results indicate that disruption of the dimer does not affect LPS or LptA binding and that monomeric LptC binds LPS and LptA at levels similar to dimeric LptC.

Dothiorelone derivatives from an endophyte Diaporthe pseudomangiferaea inhibit the activation of human lung fibroblasts MRC-5 cells

Nine new compounds (1–6 and 16–18) and nine known compounds (7–15) were isolated from Diaporthe pseudomangiferaea, an endophytic fungus obtained from the leaves of the toxic Chinese folk medicine Tylophora ouata. Their structures were elucidated by NMR spectroscopy and MS spectrometry analyses. The absolute configurations were established according to the specific rotation or electron circular dichroism method. Compounds 1, 4, 9, 11, 14 and 15 inhibited the TFG-β induced activation of human lung fibroblasts MRC-5 cells by 17.4%, 59.2%, 62.9%, 41.1%, 32.9% and 52.1% at 10 μM, respectively, while positive control pirfenidone showed 53.2% inhibition rate at 1 mM. The MTT assay showed that compounds 13 and 14 displayed cytotoxicity against BGC-823 cells, with IC50 values of 8.1 and 4.4 μM, respectively.