Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

Development of a surface plasmon resonance assay to measure the binding affinity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drug zanamivir.

Influenza is one of the most common infections of the upper respiratory tract. Antiviral drugs that are currently used to treat influenza, such as oseltamivir and zanamivir, are neuraminidase (NA) inhibitors. However, the virus may develop resistance through single-point mutations of NA. Antiviral resistance is currently monitored by a labelled enzymatic assay, which can be inconsistent because of the short half-life of the labelled product and variations in the assay conditions. In this paper, we describe a label-free surface plasmon resonance (SPR) assay for measuring the binding affinity of NA-drug interactions. Wild-type (WT) NA and a histidine 274 tyrosine (H274Y) mutant were expressed in High Five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine) was site-specifically conjugated to the 7-hydroxyl group of zanamivir, which is not involved in binding to NA, and the construct was immobilized onto a SPR sensor Chip to obtain a final immobilization response of 431 response units. Binding responses obtained for WT and H274Y mutant NAs were fitted to a simple Langmuir 1:1 model with drift to obtain the association (ka ) and dissociation (kd ) rate constants. The ratio between the binding affinities for the two isoforms was comparable to literature values obtained using labelled enzyme assays. Significant potential exists for an extension of this approach to test for drug resistance of further NA mutants against zanamivir and other antiviral drugs, perhaps paving the way for a reliable SPR biosensor assay that may replace labelled enzymatic assays.

Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance.

Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor-insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real-time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR-A). We used a scaffold approach in which anti-insulin receptor monoclonal antibody 83-7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR-A capture. The multiplex SPR system (ProteOn XPR36™, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two-site binding pattern, consistent with the literature, with a high-affinity site (dissociation constant K(D1) = 38.1 ± 0.9 nM) and a low-affinity site (K(D2) = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K(D1) = 73.2 ± 1.8 nM and K(D2) = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high-affinity site was converted to the low-affinity site. The new SPR assay enables insulin-eIR-A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling.

Anti-Fouling Properties of Phosphonium Ionic Liquid Coatings in the Marine Environment.

Biofouling is the buildup of marine organisms on a submerged material. This research tests the efficacy of phosphonium ion gels comprising phosphonium monomers ([P444VB][AOT] and [P888VB][AOT]) and free ionic liquid ([P4448][AOT], [P88814][AOT]) (10 to 50 wt%), varying copper(II) oxide biocide concentrations (0 to 2 wt%), and the docusate anion [AOT]- for added hydrophobicity. The efficacy of these formulations was tested using a seachest simulator protected from light and tidal currents in New Zealand coastal waters over the summer and autumn periods. Anti-fouling performance was correlated with the hydrophobicity of the surface (water contact angle: 14-131°) and biocide concentration. Formulations with higher hydrophobicity (i.e., less free ionic liquid and longer alkyl chain substituents) displayed superior anti-fouling performance. The presence of the copper(II) biocide negatively affected anti-fouling performance via significant increases to hydrophilicity. No correlation was observed between antimicrobial activity and anti-fouling performance. Overall, phosphonium ion gels show potential for combining anti-fouling and foul release properties.

Corticosteroid-binding globulin (CBG) reactive centre loop antibodies and surface plasmon resonance interrogate the proposed heat dependent "flip-flop" mechanism of human CBG.

Corticosteroid-binding globulin (CBG) is the predominant carrier of cortisol in circulation and is a non-inhibitory member of the serpin superfamily of serine protease inhibitors. In the stressed or "S" conformation, CBG possesses an intact exposed reactive centre loop (RCL) that can be irreversibly cleaved by elastase released from activated human neutrophils whereupon it adopts a relaxed or "R" conformation. The latter conformation has decreased affinity for cortisol, allowing the release of the majority of cortisol at sites of inflammation. Recently there has been speculation that mild increments in heat such as found in pyrexia (39-40°C) may also induce a reversible "flip-flop" of the RCL into the body of the protein structure, without cleavage, facilitating a reversible temperature-dependent release of cortisol. Here we raised a new monoclonal antibody to the RCL of human CBG and used this in concert with an existing RCL antibody and show by surface plasma resonance that, at temperatures up to 40°C, the RCL of purified CBG and the RCL of CBG in intact plasma is accessible to these two antibodies. Together, the epitopes of these antibodies span 11 consecutive amino acids (STGVTLNLTSK) of the 18 residues of the RCL. This adequate antibody cover of the RCL sequence leads to the conclusion that the proposed temperature-dependent "flip-flop" of the RCL of CBG is doubtful.

DNA G-quadruplexes show strong interaction with DNA methyltransferases in vitro.

The DNA methyltransferase enzymes (DNMTs) catalyzing cytosine methylation do so at specific locations of the genome, although with some level of redundancy. The de novo methyltransferases DNMT3A and 3B play a vital role in methylating the genome of the developing embryo in regions devoid of methylation marks. The ability of DNMTs to colocalize at sites of DNA damage is suggestive that recognition of mispaired bases and unusual structures is inherent to the function of these proteins. We provide evidence for G-quadruplex formation within imprinted gene promoters, and report high-affinity binding of recombinant human DNMTs to such DNA G-quadruplexes in vitro. These observations suggest a potential interaction of G-quadruplexes with the DNA methylation machinery, which may be of epigenetic and biological significance.

A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications.

This article describes a rapid method for purifying infectious rotavirus particles from cell culture for environmental research. The method is based on size-exclusion chromatography using TOSOH TSKgel® G5000PWXL-CP with a TSKgel® Size Exclusion G2500PWxl guard column, set up on an AKTA Explorer10. Four peaks were identified from the chromatogram and the corresponding fractions were collected and analysed by electron microscopy, 1-step quantitative reverse transcription polymerase chain reaction (RT-PCR) and qNano measurement. Infectivity potential of the recovered virus particles was determined using cell culture. Our analysis reveals that the first fraction contains majority of the intact triple-layered infectious virions while the other three fractions contain mixtures of empty capsids and intact infectious virions. Our results also indicate that there is a gross overestimation of rotaviruses in crude extracts due to encapsidated RNA in the order of 2.3 × 1011 particles and we note that estimates by qNano are similarly skewed (1.36 × 1013 particle) possibly due to empty capsids and cellular debris. In summary we present a method for purification (~12 h) of rotaviruses for a more robust and accurate quantification of virus size, surface charge and particle concentration in environmental contexts.