Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 μM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.

In this study, we annotated 49 odorant-binding proteins (OBPs) in Papilio xuthus, with four novel genes and seven improved sequences. Expression profiles identified numerous OBPs in antennae or reproductive tissues. Using two antenna-enriched general OBPs (PxutGOBP1 and PxutGOBP2) as targets, we screened three key compounds by a reverse chemical ecology strategy. Of these, an oviposition stimulant vicenin-2 could strongly interact with PxutGOBP1, representing a dissociation constant (Ki) value of 10.34 ± 0.07 μM. Molecular simulations and site-directed mutagenesis revealed the importance of His66, Thr73, and Phe118 between PxutGOBP1 and vicenin-2 interactions. Two other compounds, an ordinary floral scent β-ionone and a widely used insecticide chlorpyrifos, exhibited high affinities to PxutGOBPs (Ki < 13 μM). Furthermore, two mutations His66Ala and Thr73Ala of PxutGOBP1 significantly reduced the binding to chlorpyrifos. Our study provides insights into the putative roles of PxutGOBPs in odorant perception and identifies key binding sites of PxutGOBP1 to vicenin-2 and chlorpyrifos.

Two Ru(II) complexes, [Ru(phen)2(11-F-dppz)]2+ (Ru1, phen = 1,10-phenanthroline, 11-F-dppz = 11-fluorodipyrido[3,2-a:2\',3\'-c]phenazine) and [Ru(phen)2(11-CN-dppz)]2+ (Ru2, 11-CN-dppz = 11-cyanodipyrido[3,2-a:2\',3\'-c]phenazine), have been synthesized and characterized in this work. The binding properties of Ru1 and Ru2 with poly(A)•poly(U) RNA duplex have been investigated by spectroscopic methods and viscosity measurements. UV-vis absorption spectra and viscosity experiments demonstrate that the binding modes of Ru1 and Ru2 with poly(A)•poly(U) RNA duplex are intercalation, while the binding affinity for Ru2 is greater than that for Ru1. In addition, thermal denaturation studies reveal that both complexes significantly improve the stability of poly(A)•poly(U) duplex RNA. However, fluorescence titrations indicate that Ru1, unlike Ru2, can act as a molecular \"light switch\" for the poly(A)•poly(U) duplex RNA. The obtained results of this work indicate that the electron-withdrawing effect of substituents on the main ligands can significantly affect the binding of Ru(II) polypyridyl complexes with poly(A)•poly(U).

Glutamate transporter-1 (GLT-1) removes most glutamate in the synaptic cleft. Sulbactam confers neuronal protection against ischemic insults in the hippocampal CA1 region accompanied by the upregulation of GLT-1 expression in rats. The present study further investigates the effect of sulbactam on the binding property and uptake capacity of GLT-1 for glutamate, and the change in extracellular glutamate concentration in the hippocampal CA1 region of rats with global brain ischemia. The binding property and uptake capacity of GLT-1 were measured using a radioligand binding and uptake assay, respectively, with L-3H-glutamate. The extracellular glutamate concentration was detected using microdialysis and high-performance liquid chromatography-mass spectrometry. Neuropathological evaluation was performed based on thionin staining. It was shown that sulbactam pre-treatment changed GLT-1 binding property, including increased Bmax and decreased Kd values, increased GLT-1 uptake capacity for glutamate, and inhibited the elevation of extracellular glutamate concentration in rats with global cerebral ischemia. These effects of sulbactam were accompanied by its neuronal protection on the hippocampal CA1 neurons against delayed neuronal death resulted from ischemic insult. Furthermore, administration of GLT-1 antisense oligodeoxynucleotides, which inhibited the expression of GLT-1, blocked the aforementioned sulbactam-related effects, which suggested that GLT-1 upregulation mediated the above effect although other mechanisms independent of the upregulation of GLT-1 expression could not be excluded. It could be concluded that sulbactam improves the binding property and uptake capacity of GLT-1 for glutamate and then reduces the glutamate concentration and excitotoxicity during global cerebral ischemia, which contributes to the neuroprotection of sulbactam against brain ischemia.

Hemoglobin (Hb) is the most abundant protein in the blood. It is vital for the living as oxygen carriers. Some of the very pure Hb-containing biological fluids are currently under clinical trial. However, the removal and purification of Hb from the blood are quite difficult, especially when it is at a low concentration level. In this study, the molecularly imprinted polymeric nanoparticles (MIPNs) were prepared using N-methacryloyl-histidine methyl ester (MAH) by mini-emulsion polymerization technique for specific binding of human hemoglobin (HHb). MIPNs in monosize form have a size of 152 ± 4 nm. They also have a high binding capacity (32.33 mg/g) of HHb. MIPNs retain 84% of the re-binding capacity for HHb after 10 cycles. The nanoparticles have 16 and 5 times higher binding capacity of HHb, respectively, in the presence of bovine serum albumin and lysozyme. Thanks to their high binding capacity and selectivity, MIPNs will allow them to be detected selectively for different target molecules. According to molecular docking, the main binding forces depend on hydrogen bonds and Van der Waals forces in the interaction within 5 Å around MAH molecule are observed through the amino acid residues of HHb at β1 and β2 subunit. The statistical mechanical analysis of docking showed that the free energy (ΔG) is -2732.14 kcal/mol, which indicates the interaction between MAH and HHb is energetically favorable at 298.15°K.

BACKGROUND: In the drug discovery field, the binding affinities and binding kinetics of drug candidates are very important. Angiotensin II type 1 (AT1) receptor antagonists, e.g., candesartan, telmisartan, irbesartan, losartan and valsartan, show high affinities and long-lasting bindings to the receptor, making them preferred medications for hypertension treatment. However, the molecular binding properties of AT1 receptor antagonists are controversial.
METHODS: In this work, we established a profile to study the phenotypic properties of AT1 receptor antagonists with label-free dynamic mass redistribution (DMR) assays in native human cells. With noninvasive features, DMR assay were conducted in multiple formats. Eleven antagonists were systematically evaluated with angiotensin II as an agonist probe in the Hep G2 cell line, which endogenously expresses the AT1 receptor.
RESULTS: The IC50 values to the AT1 receptor of individual antagonist varied with different incubation times. The antagonists showed competitive behavior with angiotensin II. Schild analysis was used to analyze the competitive behavior of the antagonist. All of the antagonist showed long-lasting possession of the AT1 receptor, except telmisartan. The systematic evaluation of the antagonists implied that 11 antagonists showed high binding affinity but distinct binding modes to AT1 receptor.
CONCLUSIONS: This study demonstrated that the DMR assay has great potential for determining the pharmacological parameters of ligands. This work may serve as guidance for other receptor and ligand assays.

In this work, two Ru(II)-dppz (dppz = dipyrido[3,2-a:2\',3\'-c]phenazine) complexes containing fluorine substituents, [Ru(bpy)2(7-F-dppz)]2+ (Ru1, bpy = 2,2\'-bipyridine, 7-F-dppz = 7-fluorodipyrido[3,2-a:2\',3\'-c]phenazine) and [Ru(phen)2(7-F-dppz)]2+ (Ru2, phen = 1,10-phenanthroline), have been synthesized and characterized. Binding properties of Ru1 and Ru2 with the RNA poly(U)·poly(A) ∗ poly(U) triplex have been studied by spectroscopic methods and viscosity measurements. The obtained results indicate that the binding differences of the two complexes with the triplex may be attributed to the ancillary ligand effects, implying that the better planarity and greater hydrophobicity of ancillary ligands are advantageous to the π-π stacking interaction between Ru2 and the triplex, thus Ru2 stabilizes the triplex strongly than Ru1. Denaturation of the triplex shows that both Ru1 and Ru2 can not only highly stabilize the template duplex of the triplex, but also significantly stabilize the third strand. Compared with the triplex stabilizing effects for the reported Ru(II)-dppz complexes, thermal melting experiments suggest that the fluorine substituent on the ligand dppz can probably decrease electrostatic repulsion between the three strands of the triplex, thereby Ru1 and Ru2 significantly increase the triplex stabilization. Results obtained from this work further confirm that the substituent electron effect of dppz-based ligands and the planarity and hydrophobicity of ancillary ligands play an important role in the triplex stabilizing effects by Ru(II)-dppz complexes.

In order to explore Qingke β-glucans as functional food ingredients for prevention of obesity, the physicochemical structures, in vitro binding properties, and inhibitory activities on pancreatic lipase of β-glucans from three different Qingke cultivars, including Ganyucang (black), Dingqing (blue), and Zangqing 320 (white), were investigated and compared. Results showed that molecular weights, particle sizes, and intrinsic viscosities of β-glucans from colored (black and blue) Qingke cultivars were much higher than those of white Qingke β-glucans, respectively. In addition, the constituent monosaccharides of β-glucans from colored Qingke cultivars were determined as arabinose, xylose, glucose, and galactose, and glucose was the dominant monosaccharide. Furthermore, colored Qingke β-glucans exerted strong fat binding, cholesterol binding, and bile-acid binding capacities, as well as inhibitory activities on pancreatic lipase, which were much higher than those of white Qingke β-glucans. Indeed, the fat binding, cholesterol binding, and bile-acid binding capacities, as well as the inhibitory activities on pancreatic lipase of Qingke β-glucans were positively associated with their molecular weights and intrinsic viscosities. Results are beneficial for better understanding of the structure-function relationship of Qingke β-glucans, and β-glucans from colored Qingke cultivars (Ganyucang and Dingqing) could be further explored as functional food ingredients for prevention of obesity.

As the first biochemical step of olfactory reception and recognition, odorant binding proteins (OBPs) have been demonstrated to be essential. Considering functional diversities of OBPs within a single species, we here extended the characterization of two other OBPs from Spodoptera exigua, belonging to insect Classic OBPs. With a combination of transcriptome and Rapid Amplification of cDNA End (RACE) approaches, two OBP genes in S. exigua were identified, namely SexiOBP1 and OBP7. Expression pattern analysis revealed that both of them exhibited a distinct expression pattern, where OBP1 was broadly and highly expressed in several tissues including antennae of adults whereas OBP7 was abundant only in the antennae of both sexes, strongly indicative of olfactory roles. Further, binding assays showed that the two SexiOBPs shared a common odorant-response spectrum with considerable affinities to host odorants of acetophenone, farnesol and β-ionone (Ki<20μM). Specially, OBP1 could strongly bind an insect attractant β-caryophyllene (Ki=2.76μM) released by maize. Intriguingly, the major sex pheromone of S. exigua, Z9,E12-14:Ac, was the best ligand for OBP7 with Ki value of 7.58μM. Ligand structural analysis revealed that the two SexiOBPs were capable of accommodating different types of ligands in shape and size, possibly implying the plasticity of binding pockets. Ultimately, comparison of binding properties among 10 SexiOBPs including the two OBPs in this study implied a cross-talk in functions, i.e. different OBPs are also suitable to accept some common odorants except for unique ligands. Taken together, this study has provided evidence for their involvements in seeking and orientation of host plants, and meanwhile indicates functional conservation and differences between OBP1 and OBP7 from S. xigua.