Neutron scattering: A versatile tool for probing complex solutions and surfaces

Neutron scattering has significant benefits for examining the structure of complex samples. Cold (slow) neutrons are non-damaging and predominantly interact with the atomic nucleus, meaning that they can penetrate deeply into samples, which allows for flexibility in the design of samples studied. Most importantly, there is a strong difference in neutron scattering length (i.e., scattering power) between protium ((_1^1)H, 99.98% natural abundance) and deuterium ((_1^2)H, or D, 0.015%). Through the mixing of H2O and D2O in the samples and in some cases the deuterium labelling of the biomolecules, components within a complex can be hidden or enhanced in the scattering signal. This enables both the overall structure and the relative distribution of components within a complex to be resolved. In this talk I will discuss the technical premise behind performing neutron scattering experiments and highlight examples where neutron scattering has been used to examine complex bio-pharmaceutical related phenomena. Examples will include in situ chromatography measurements and complex and realistic bio-mimetics at buried interfaces

The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. We have examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers, using a combination of surface pressure measurements, external reflection FTIR spectroscopy and neutron reflectometry. Results highlighted differences in the protein binding mechanisms, and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity

Adsorption of sodium hexanoate on α-alumina.

Neutron reflection and adsorption isotherm measurements have been used to study the adsorption behaviour of hexanoic acid onto α-alumina surfaces. Importantly, the pH dependence of the behaviour has been characterised with a pronounced maximum in adsorption identified at a pH of approximately 5, close to the pKa of the acid. The adsorbed layer is identified as a bilayer, which is reasonable given the hydrophilic nature of both side of the layer, and has a thickness of 13 Å, suggesting significant extent of interdigitation. At pH 5, the layer has much lower extent of hydration relative to the higher pH of 7, consistent with the increased total adsorption at pH 5. A number of different mechanisms for the binding of the hexanoic acid to the surface are considered. The experimental data, combined with calculations using equilibrium/binding constants of the surface and ligands, indicates that a ligand exchange reaction may be the most significant mechanism.This is the author's accepted manuscript. The final version has been published by Elsevier in the Journal of Colloid and Interface Science here: http://www.sciencedirect.com/science/article/pii/S0021979713005560

Tryptophan to arginine substitution in puroindoline b alters binding to model eukaryotic membranes

We have studied how puroindoline-b (PINB) mutants bind to model eukaryotic membranes dependent on binary composition of anionic:zwitterionic phospholipids and the presence of cholesterol and sphingomyelin in the model membrane. We have found that the trends in lipid binding behavior are different for wild-type PINB compared to its naturally occurring PINB(Trp44Arg) mutant form and have seen evidence of protein-induced domain formation within the lipid layer structure. Results show that selective binding of antimicrobial peptides to different membrane types is as a result of differences in lipid composition and the arrangement of lipids within the membrane surface. However, membrane-binding behavior is not easily predicted; it is determined by net charge, hydrophobicity, and the amphiphilicity of the protein/peptide lipid-binding domain

Strategies towards improving pharmacological management of asthma during pregnancy

Maternal asthma represents a significant burden to individuals and the healthcare system, affecting 1 in 10 pregnancies worldwide. Approximately 50% of asthmatic women experience a deterioration of asthma control at some stage during pregnancy, with a number requiring use of oral corticosteroids for the management of acute exacerbations. The presence of maternal asthma and exacerbations during pregnancy is a noted risk factor for a range of adverse perinatal outcomes including preterm birth, small-for-gestational age, pre-eclampsia, and gestational diabetes. These negative impacts highlight the need for evidence-based approaches for improving asthma management during pregnancy and subsequent perinatal outcomes. Despite this, relatively small progress has been made in enhancing the management of maternal asthma in the clinical setting. A major challenge in improving outcomes of asthmatic pregnancies is that there is no single simplified approach for improving outcomes, but rather the requirement to consider the dynamic relationship between a myriad of interrelated factors that ultimately determine an individual's ability to maintain adequate asthma control. Understanding how these factors are impacted by pregnancy and how they can be addressed through various interventions is therefore important in optimising health outcomes. This review summarises key factors involved in influencing outcomes associated with maternal asthma. This includes an overview of the use of asthma medications in pregnancy, while also considering the impacts of interrelated aspects such as medication adherence, health-seeking behaviours, biological and lifestyle factors, co-morbidities, and asthma self-management strategies on asthma control. Addressing such factors through multidisciplinary approaches towards treatment have potential to improve the health of mothers and their offspring. Optimising asthma control should be a high priority within the antenatal setting, with women advised about the importance of good asthma control, managing asthma actively throughout pregnancy by utilising their asthma medications, and managing exacerbations in a timely and effective manner

A comparison of didodecyldimethylammonium bromide adsorbed at mica/water and silica/water interfaces using neutron reflection.

The layer structure of the dichain alkyl ammonium surfactant, didodecyldimethylammonium bromide (DDAB), adsorbed from water on to silica and mica surfaces has been determined using neutron reflection. Although sometimes considered interchangeable surfaces for study, we present evidence of significant differences in the adsorbed layer structure below the critical micelle concentration. A complete DDAB bilayer was assembled at the water/mica interface at concentrations below the critical micelle concentration (CMC). In contrast it is not until the CMC was reached that the complete bilayer structure formed on the oxidised silicon crystal. Removal of the complete bilayer on both surfaces was attempted by both washing and ion exchange yet the adsorbed structure proved tenacious.BPThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jcis.2016.06.01

A multipronged approach to understanding the form and function of hStaufen protein

Staufen is a dsRNA binding protein that plays an essential role in many aspects of RNA regulation, such as mRNA transport, Staufen-mediated mRNA decay and the regulation of mRNA translation. Staufen is a modular protein characterized by the presence of conserved consensus amino acid sequences that fold into double-stranded RNA binding domains (RBDs) as well as degenerated RBDs that maintain the α-β-β-β-α fold but are unable to bind RNA and are instead involved in protein-protein interactions. The variety of biological processes in which Staufen participates in the cell suggests that this protein associates with many diverse RNA targets, some of which have been identified experimentally. Staufen binding mediates the recruitment of effectors via protein-protein and protein-RNA interactions. The structural determinants of a number of these interactions, as well as the structure of full-length Staufen, remain unknown. Here, we present the first solution structure models for full-length human Staufen155, showing that its domains are arranged as beads-on-a-string in the absence of RNA