Binding Spectra Research Articles

A theoretical analysis of single molecule protein sequencing via weak binding spectra.

We propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding (Havranek, et al., 2013) to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to weakly-binding probes with dissociation constants of tens or hundreds of micromolar, and bypasses photobleaching limitations associated with other fluorescence-based approaches to protein sequencing. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.

Protein and RNA dynamical fingerprinting

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons

Context: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities.Objective: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined.Materials and methods: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h.Results: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2.Discussion and conclusions: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.

Global Identification of HIF-1α Target Genes in Benzene Poisoning Mouse Bone Marrow Cells.

Benzene is a hematopoietic toxicant, and hematopoietic cells in bone marrow (BM) are one of the main targets for its action, especially hematopoietic stem cells (HSCs). Hypoxia-inducible factor-1α (HIF-1α) is associated with the metabolism and physiological functions of HSCs. We previously found that the mechanism of regulation of HIF-1α is involved in benzene-induced hematopoietic toxicity. In this study, chromatin immunoprecipitation sequencing (ChIP-Seq) technologies were used to analyze the genome-wide binding spectrum of HIF-1α in mouse BM cells, and specific HIF-1α target genes and pathways associated with benzene toxicity were screened and validated. By application of the ChIP-Seq technique, we identified target genes HIF-1α directly binds to and regulates. Forty-two differentially down-regulated genes containing the HIF-1α specific binding site hypoxia response element (HRE) were found, of which 25 genes were with biological function. Moreover, the enrichment analysis of signal pathways indicated that these genes were significantly enriched in the Jak-STAT signaling pathway, Natural killer cell mediated cytotoxicity, the Fc epsilon RI signaling pathway, Pyrimidine metabolism, the T cell receptor signaling pathway, and Transcriptional misregulation in cancer. After verification, 11 genes involved in HSC self-renewal, cell cycle, differentiation, and apoptosis pathways were found to be significantly reduced, and may participate in benzene-induced hematotoxicity. Our study provides a new academic clue for the mechanism of benzene hematotoxicity.

Distinct binding affinities of odorant-binding proteins from the natural predator Chrysoperla sinica suggest different strategies to hunt prey

Chrysoperla sinica is an important natural predator of many notorious agricultural pests. Understanding its olfactory mechanism can help enhance the effectiveness of C. sinica in biological control. In the present study, we investigated the tissue expression patterns of 12 odorant-binding protein (OBP) genes from C. sinica (CsinOBPs). The results of quantitative real-time polymerase chain reaction (qPCR) showed that CsinOBP1, CsinOBP2, CsinOBP3, CsinOBP4, CsinOBP6, CsinOBP7, CsinOBP9, CsinOBP10, and CsinOBP12 were predominantly expressed in the antennae of both sexes, indicating their roles in olfaction. Additionally, the qPCR analysis revealed that the 12 CsinOBP genes had distinct expression patterns, while the motif-pattern investigation suggested that the OBPs had different ligands. The ligand-binding assay showed that CsinOBP1 and CsinOBP10 had broader binding spectra than did the other OBPs. Thus, CsinOBP1 was able to bind not only plant volatiles (such as farnesol, cis-3-hexenyl hexanoate, geranylacetone, β-ionone, 2-tridecanone, and trans-nerolidol) but also the aphid alarm pheromone (E)-β-farnesene. On the other hand, CsinOBP2 and CsinOBP6 exhibited relatively narrow binding spectra, only binding ethyl benzoate. The study also identified several compounds that can potentially be used to develop slow-release agents attracting C. sinica and to improve search strategies for insect pest control.

Expression patterns and ligand binding characterization of Plus-C odorant-binding protein 14 from Adelphocoris lineolatus (Goeze)

Odorant-binding proteins (OBPs) can bind and transport hydrophobic odorants across the sensillum lymph to the olfactory receptors (ORs) and play crucial roles in insect chemosensory systems. Although the ligand spectra of classical OBPs have been extensively characterized, little is known about OBPs in the Plus-C subgroup. Here, we focus on AlinOBP14, a Plus-C OBP from the hemipteran mirid bug pest Adelphocoris lineolatus (Goeze). Quantitative real-time PCR experiments suggest that AlinOBP14 is ubiquitously expressed at different developmental stages but is highly expressed in the adult head, the non-chemosensory organ. Fluorescence-based competitive binding assays show that β-ionone, nerolidol, farnesol and insect juvenile hormone III (JHIII) strongly bind to AlinOBP14. No significant internal binding pocket is predicted by homology modeling. Instead, the long N-terminal and C-terminal regions and parts of several α-helixes form a cupped cavity to accommodate ligands. Molecular docking reveals that the four potential ligands have distinct binding orientations, implying different roles of the N-terminal extension in ligand recognition. This hypothesis is further confirmed via a ligand binding assay in which the recombinant N-terminal mutant AlinOBP14 displays comparable binding affinities for β-ionone and trans, trans-farnesol but decreased binding affinities for nerolidol and JHIII. Thus, our current study is the first to characterize the ligand binding spectra of a Plus-C OBP in hemipteran insect species and reveals that N-terminal extensions could be required for its recognition of putative ligands.

Ab Initio Description of Clustering Phenomena in Spectra of Light Nuclei

An approach has been presented to take into account clustering properties of nuclei by the introduction of cluster components in the basis used in microscopic ab initio calculations and to calculate the cluster characteristics of nuclei by the projection of the A-nucleon wavefunction on wavefunctions of cluster channels. The binding energy, spectra, and cluster spectroscopic factors of the α + t channel have been calculated for the ground and lower excited states of the 7Li nucleus. It has been shown that, except for a single state these states are strongly clustered, which is in qualitative agreement with spectroscopic data. It has also been found that the contribution of “noncluster” components to the binding energy is very large even in strongly clustered states.

Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge.

Broadly cross-reactive antibodies (Abs) that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes, including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (MAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the MAbs bound HAs from multiple strains of group 1 viruses, and one MAb, 05-2G02, bound to both group 1 and group 2 influenza A virus HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two MAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One MAb, 70-1F02, cocrystallized with H5 HA and showed heavy-chain-only interactions similar to those seen with the previously described CR6261 anti-stalk antibody. Finally, we show that antibodies that compete with these MAbs are prevalent in serum from an individual recently infected with the A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections by zoonotic or emerging pandemic influenza viruses.IMPORTANCE The rise in zoonotic infections of humans by emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available antiviral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved "stalk" domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus.

The Odorant Binding Protein 6 Expressed in Sensilla Chaetica Displays Preferential Binding Affinity to Host Plants Volatiles in Ectropis obliqua.

The monophagous tea geometrid Ectropis obliqua selectively feed on tea plants, requiring the specialized chemosensory system to forage for certain host. A deep insight into the molecular basis would accelerate the design of insect-behavior-modifying stimuli. In the present study, we focused on the odorant-binding protein 6 (EoblOBP6) with the high abundance in legs transcriptome of E. obliqua moths. qRT-PCR coupled with western blot analyses revealed the dual expression pattern of EoblOBP6 in antennae and legs. Cellular immunolocalization indicated that EoblOBP6 was predominantly labeled in the outer sensillum lymph of uniporous sensilla chaetica, which is not innervated by sensory neurons. No specific staining was observed in other sensillum types. The fluorescence competition assay showed a relatively narrow binding spectrum of recombinant EoblOBP6. EoblOBP6 could not only bind with intact tea plant volatiles benzaldehyde but also display high binding ability to nerolidol and α-farnesene which are tea plant volatiles dramatically induced by herbivore infestation. Besides, EoblOBP6 tightly bound to the aversive bitter alkaloid berberine. Taken together, EoblOBP6 displayed an unusual expression in sensilla chaetica, exhibited the potential involvement in olfaction and gustation, and may play a functional role in host location of female E. obliqua moths.

Clostridium perfringens Virulent Bacteriophage CPS2 and Its Thermostable Endolysin LysCPS2.

Clostridium perfringens is one of the most common causes of food-borne illness. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Here, we isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken feces. The CPS2 phage contains a 17,961 bp double-stranded DNA genome with 25 putative ORFs, and belongs to the Picovirinae, subfamily of Podoviridae. Bioinformatic analysis of the CPS2 genome revealed a putative endolysin, LysCPS2, which is homologous to the endolysin of Clostridium phage phiZP2 and phiCP7R. The enzyme showed strong lytic activity against C. perfringens with optimum conditions at pH 7.5–10, 25–65 °C, and over a broad range of NaCl concentrations. Interestingly, LysCPS2 was found to be highly thermostable, with up to 30% of its lytic activity remaining after 10 min of incubation at 95 °C. The cell wall binding domain in the C-terminal region of LysCPS2 showed a binding spectrum specific to C. perfringens strains. This is the first report to characterize highly thermostable endolysin isolated from virulent C. perfringens bacteriophage. The enzyme can be used as an alternative biocontrol and detection agent against C. perfringens.

Inhibitory effect of α-terpinyl acetate on cytochrome P450 2B6 enzymatic activity

Human cytochrome P450 2B6 is an important hepatic enzyme for the metabolism of xenobiotics and clinical drugs. Recently, more attention has been paid to P450 2B6 because of the increasing number of drugs it metabolizes. It has been known to interact with terpenes, the major constituents of the essential oils used for various medicinal purposes. In this study, the effect of monoterpenes on P450 2B6 catalytic activity was investigated. Recombinant P450 2B6 was expressed in Escherichia coli and purified using Ni-affinity chromatography. The purified P450 2B6 enzyme displayed bupropion hydroxylation activity in gas-mass spectrometry (GC-MS) analysis with a kcat of 0.5 min−1 and a Km of 47 μM. Many terpenes displayed the type I binding spectra to purified P450 2B6 enzyme and α-terpinyl acetate showed strong binding affinity with a Kd value of 5.4 μM. In GC-MS analysis, P450 2B6 converted α-terpinyl acetate to a putative oxidative product. The bupropion hydroxylation activity of P450 2B6 was inhibited by α-terpinyl acetate and its IC50 value was 10.4 μM α-Terpinyl acetate was determined to be a competitive inhibitor of P450 2B6 with a Ki value of 7.6 μM. The molecular docking model of the binding site of the P450 2B6 complex with α-terpinyl acetate was constructed. It showed the tight binding of α-terpinyl acetate in the active site of P450 2B6, which suggests that it could be a competitive substrate for P450 2B6.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

ABSTRACTSome viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2, *3, *4, and *10

ABSTRACTHumans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower kcat values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased Km values and diminished kcat values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.

Biochemical and biophysical insights into the metal binding spectrum and bioactivity of arginase of Entamoeba histolytica.

The human protozoan pathogens possess the essential metalloenzyme arginase (Arg) which catalyses the catabolism of l-arginine to l-ornithine and urea. This being the first committed step in polyamine biosynthesis is a potential drug target for protozoan diseases. In pathogenic organisms, arginase plays a crucial role in depleting host l-arginine, a substrate for nitric oxide synthase (NOS) that participates in protective immunity, thereby evading host immune response. In this study, the metal binding spectrum of EhArg has been determined. This study focuses on the biochemical and biophysical characterization of arginase from Entamoeba histolytica (EhArg), majorly characterizing the bivalent metal selectivity and metal binding kinetics of purified EhArg using Surface Plasmon Resonance and inductively coupled plasma mass spectroscopy. Investigation of the active site chemistry and total metal content using molecular docking and ICP-MS unraveled the fact that two Mn2+ ions are required for the enzyme to be fully functional. However, chelating loosely bound Mn2+ and replacing it with a variety of bivalent metal ions including Mg2+, Zn2+, Ni2+, Hg2+, Cu2+, Co2+, Ca2+ and Cd2+ retains its enzymatic activity. Further, the role of nine bivalent ions in the activation of EhArg was studied thermodynamically and biochemically. Phylogenetic and sequence analysis and oligomerization studies of EhArg show that unlike other eukaryotic arginases, EhArg exists in monomeric and dimeric form in solution and shows the highest similarity with bacterial arginase. This study unveiled interesting facts about EhArg that the enzyme has evolved to utilize available metal ion cofactors and survive the inhospitable environment within the host.

Two‐Photon Optical Properties in Individual Organic–Inorganic Perovskite Microplates

AbstractMetal‐halide perovskites are recently extensively investigated as light absorbing material in solar cells. The outstanding optoelectronic properties and tunable light emission of the perovskites also make them promising candidates for light emitting diodes and lasers. However, understanding the relevant mechanisms and processes of the dependence of perovskite light emission on temperature and crystal size is still challenging. Herein, the CH3NH3PbBr3 monocrystals of different sizes are uniformly excited by two‐photon absorption at 800 nm (100 fs, 1 KHz). In contrast to the reported relative large exciton binding energy (≈76 meV) and spectrum clearly resolved excitonic absorption, the light emission origin in CH3NH3PbBr3 microcrystals at room temperature is unambiguously determined to be dominated by free electron–hole bimolecular recombination. The coherent light emission threshold of CH3NH3PbBr3 microcrystal increases with temperature, which is closely related to the temperature induced transition from exciton gas to free charge carriers. In addition, the coherent light emission threshold is found to decrease with the microcrystal size, which could be well interpreted by the interaction between the optical confinement, defect density, and cavity quantum electrodynamics effect. These results presented here may facilitate the development of perovskite light emitting diodes and lasers.

Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles

The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.

Structure-Guided Functional Annotation of the Influenza A Virus NS1 Protein Reveals Dynamic Evolution of the p85β-Binding Site during Circulation in Humans.

ABSTRACTRational characterization of virulence and host-adaptive markers in the multifunctional influenza A virus NS1 protein is hindered by a lack of comprehensive knowledge about NS1-host protein protein interfaces. Here, we surveyed the impact of amino acid variation in NS1 at its structurally defined binding site for host p85β, a regulator of phosphoinositide 3-kinase (PI3K) signaling. Structure-guided alanine scanning of all viral residues at this interface defined 10 positions contributing to the interaction, with residues 89, 95, 98, 133, 145, and 162 being the most important. A bioinformatic study of >24,000 publicly available NS1 sequences derived from viruses infecting different hosts highlighted several prevalent amino acid variants at the p85β interface that either enhanced (I95) or weakened (N135, T145, L161, Y161, S164) p85β binding. Interestingly, analysis of viruses circulating in humans since the 1918 pandemic revealed the temporal acquisition of functionally relevant variants at this interface. I95 (which enhanced p85β binding) quickly became prevalent in the 1940s and experimentally conferred a fitness advantage to a recombinant 1930s-based H1N1 virus in human lung epithelial cells. Surprisingly, H1N1 and H3N2 viruses recently acquired T145 or N135, respectively, which diminished p85β binding but apparently not the overall fitness in the human population. Evolutionary analyses revealed covariation of the NS1-p85β binding phenotype in humans with functional changes at multiple residues in other viral proteins, suggesting an unexplored compensatory or synergistic interplay between phenotypes in vivo. Overall, our data provide a resource to understand the consequences of the NS1-p85β binding spectrum of different influenza viruses and highlight the dynamic evolution of this property in viruses circulating in humans.IMPORTANCE In humans, influenza A viruses are responsible for causing seasonal epidemics and occasional pandemics. These viruses also circulate and evolve in other animal species, creating a reservoir from which novel viruses with distinct properties can emerge. The viral nonstructural protein NS1 is an important host range determinant and virulence factor that exhibits strain-specific interactions with several host factors, although few have been characterized extensively. In the study described here, we comprehensively surveyed the impact of natural and unnatural NS1 variations on the binding of NS1 to host p85β, a subunit of phosphoinositide 3-kinase that regulates intracellular metabolism and contributes to virus replication and virulence. We define the p85β-binding site on NS1 and provide a predictive resource to assess this ability of NS1 in viruses from different hosts. Strikingly, we uncover a spectrum of p85β binding by different NS1 proteins and reveal that viruses evolving in humans have undergone dynamic changes in this NS1 function over the last century.

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b5 enhanced the kcat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min−1) with little effect on KM (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.

Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity.

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

Tissue distribution and functional characterization of odorant binding proteins in Chilo suppressalis (Lepidoptera: Pyralidae)

Odorant binding proteins (OBPs) play important roles in the insect olfaction and other diverse physiological processes. Forty OBP genes have been molecularly identified from Chilo suppressalis (Walker), a notorious rice pest in Asian countries, but little is known about the olfactory function for most of these genes. In the present study, we first determined the tissue expression profiles of 34 OBPs (excluding two general odorant bonding proteins (GOBPs) and four pheromone binding protein (PBPs)) by quantitative real-time PCR (qPCR), and found that 9 genes (OBP1, 3, 4, 11, 15, 17, 19, 20 and 24) were specifically or predominantly expressed in antennae of both sexes, suggesting their roles in olfaction, while three genes (OBP29, 30 and 32) were almost not expressed in antennae. Focusing on olfactory roles, the ligand specificities of six antenna specifically or predominantly expressed genes were further investigated for 35 plant volatiles, using the fluorescence competitive binding assays. The results revealed that six OBPs displayed different ligand preference, suggesting a differentiation of OBPs in ligand binding spectrum. Of six tested OBPs, OBP3, 11, 17, 19 and 31 showed moderate (Ki=10.21–19.85μM) or high (Ki<10.00μM) binding affinity for 11 and one plant volatiles, respectively. In particular, a plant volatile β-ionone had high or moderate binding to all five OBPs. Our study suggests that these five OBP genes play important roles in the perception of different host plant volatiles, providing insight into the olfactory mechanism in C. suppressalis.