The role of protein hydrophobicity in thionin-phospholipid interactions: a comparison of α1 and α2-purothionin adsorbed anionic phospholipid monolayers.

The plant defence proteins α1- and α2-purothionin (Pth) are type 1 thionins from common wheat (Triticum aestivum). These highly homologous proteins possess characteristics common amongst antimicrobial peptides and proteins, that is, cationic charge, amphiphilicity and hydrophobicity. Both α1- and α2-Pth possess the same net charge, but differ in relative hydrophobicity as determined by C18 reversed phase HPLC. Brewster angle microscopy, X-ray and neutron reflectometry, external reflection FTIR and associated surface pressure measurements demonstrated that α1 and α2-Pth interact strongly with condensed phase 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) monolayers at the air/liquid interface. Both thionins disrupted the in-plane structure of the anionic phospholipid monolayers, removing lipid during this process and both penetrated the lipid monolayer in addition to adsorbing as a single protein layer to the lipid head-group. However, analysis of the interfacial structures revealed that the α2-Pth showed faster disruption of the lipid film and removed more phospholipid (12%) from the interface than α1-Pth. Correlating the protein properties and lipid binding activity suggests that hydrophobicity plays a key role in the membrane lipid removal activity of thionins.

Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications.

In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.

Introducing structural flexibility into porphyrin-DNA zipper arrays.

A more flexible nucleotide building block for the synthesis of new DNA based porphyrin-zipper arrays is described. Changing the rigid acetylene linker between the porphyrin substituent and the 2'-deoxyuridine to a more flexible propargyl amide containing linkage leads in part to an increased duplex stability. The CD spectra reveal different electronic interactions between the porphyrins depending on the type of linker used. Molecular modelling suggests large variation of the relative orientation of the porphyrins within the major groove of the DNA. The porphyrins can be metallated post-synthetically with different metals as shown with zinc, cobalt and copper. The spectroscopic features do not alter drastically upon metallation apart from the CD spectra, and the stability of the metal complex is highly dependent on the nature of the metal. As shown by CD spectroscopy, the zinc porphyrin is rapidly demetallated at high temperatures. Globular structure determination using SAXS indicates that a molecular assembly comprised of a two to four helical bundle dominates in solution at higher concentrations (≥50 µM) which is not observed by spectroscopy at lower concentrations (≤1 µM).

Lipid binding interactions of antimicrobial plant seed defence proteins: puroindoline-a and ß-purothionin.

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and ß-purothionin (ß-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and ß-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and ß-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. ß-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and ß-Pth hydrophobic regions.

Puroindoline-a, a lipid binding protein from common wheat, spontaneously forms prolate protein micelles in solution.

The self-assembly in solution of puroindoline-a (Pin-a), an amphiphilic lipid binding protein from common wheat, was investigated by small angle neutron scattering, dynamic light scattering and size exclusion chromatography. Pin-a was found to form monodisperse prolate ellipsoidal micelles with a major axial radius of 112 ± 4.5 Å and minor axial radius of 40.4 ± 0.18 Å. These protein micelles were formed by the spontaneous self-assembly of 38 Pin-a molecules in solution and were stable over a wide pH range (3.5-11) and at elevated temperatures (20-65 °C). Pin-a micelles could be disrupted upon addition of the non-ionic surfactant dodecyl-ß-maltoside, suggesting that the protein self-assembly is driven by hydrophobic forces, consisting of intermolecular interactions between Trp residues located within a well-defined Trp-rich domain of Pin-a.

Mapping open knowledge institutions: an exploratory analysis of Australian universities.

While the movement for open research has gained momentum in recent years, there remain concerns about the broader commitment to openness in knowledge production and dissemination. Increasingly, universities are under pressure to transform themselves to engage with the wider community and to be more inclusive. Open knowledge institutions (OKIs) provide a framework that encourages universities to act with the principles of openness at their centre; not only should universities embrace digital open access (OA), but also lead actions in cultivating diversity, equity, transparency and positive changes in society. This leads to questions of whether we can evaluate the progress of OKIs and what are potential indicators for OKIs. As an exploratory study, this article reports on the collection and analysis of a list of potential OKI indicators. Data for these indicators are gathered for 43 Australian universities. The indicators provide high-dimensional and complex signals about university performances. They show evidence of large disparities in characteristics such as Indigenous employment and gender equity, and a preference for repository-mediated OA across Australian universities. We demonstrate use of the OKI evaluation framework to categorise these indicators into three platforms of diversity, communication and coordination. The analysis provides new insights into the Australian open knowledge landscape and ways of mapping different paths of OKIs.

MyCites: a proposal to mark and report inaccurate citations in scholarly publications.

BACKGROUND: Inaccurate citations are erroneous quotations or instances of paraphrasing of previously published material that mislead readers about the claims of the cited source. They are often unaddressed due to underreporting, the inability of peer reviewers and editors to detect them, and editors' reluctance to publish corrections about them. In this paper, we propose a new tool that could be used to tackle their circulation. METHODS: We provide a review of available data about inaccurate citations and analytically explore current ways of reporting and dealing with these inaccuracies. Consequently, we make a distinction between publication (i.e., first occurrence) and circulation (i.e., reuse) of inaccurate citations. Sloppy reading of published items, literature ambiguity and insufficient quality control in the editorial process are identified as factors that contribute to the publication of inaccurate citations. However, reiteration or copy-pasting without checking the validity of citations, paralleled with lack of resources/motivation to report/correct inaccurate citations contribute to their circulation. RESULTS AND DISCUSSION: We propose the development of an online annotation tool called "MyCites" as means with which to mark and map inaccurate citations. This tool allows ORCID users to annotate citations and alert authors (of the cited and citing articles) and also editors of journals where inaccurate citations are published. Each marked citation would travel with the digital version of the document (persistent identifiers) and be visible on websites that host peer-reviewed articles (journals' websites, Pubmed, etc.). In the future development of MyCites, challenges such as the conditions of correct/incorrect-ness and parties that should adjudicate that, and, the issue of dealing with incorrect reports need to be addressed.

Evaluating the impact of open access policies on research institutions.

The proportion of research outputs published in open access journals or made available on other freely-accessible platforms has increased over the past two decades, driven largely by funder mandates, institutional policies, grass-roots advocacy, and changing attitudes in the research community. However, the relative effectiveness of these different interventions has remained largely unexplored. Here we present a robust, transparent and updateable method for analysing how these interventions affect the open access performance of individual institutes. We studied 1,207 institutions from across the world, and found that, in 2017, the top-performing universities published around 80-90% of their research open access. The analysis also showed that publisher-mediated (gold) open access was popular in Latin American and African universities, whereas the growth of open access in Europe and North America has mostly been driven by repositories.

Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex.

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process.

Thermal equivalence of DNA duplexes for probe design.

We present the theory of thermal equivalence in the framework of the Peyrard-Bishop model and some of its anharmonic variants. The thermal equivalence gives rise to a melting index τ which maps closely the experimental DNA melting temperatures for short DNA sequences. We show that the efficient calculation of the melting index can be used to analyse the parameters of the Peyrard-Bishop model and propose an improved set of Morse potential parameters. With this new set we are able to calculate some of the experimental melting temperatures to ± 1.2 °C. We review some of the concepts of sequencing probe design and show how to use the melting index to explore the possibilities of gene coverage by tuning the model parameters.

Anharmonic behavior in the multisubunit protein apoferritin as revealed by quasi-elastic neutron scattering.

Quasi-elastic neutron scattering (QENS) has been used to study the deviation from Debye-law harmonic behavior in lyophilized and hydrated apoferritin, a naturally occurring, multisubunit protein. Whereas analysis of the measured mean squared displacement (msd) parameter reveals a hydration-dependent inflection above 240 K, characteristic of diffusive motion, a hydration-independent inflection is observed at 100 K. The mechanism responsible for this low-temperature anharmonic response is further investigated, via analysis of the elastic incoherent neutron scattering intensity, by applying models developed to describe side-group motion in glassy polymers. Our results suggest that the deviation from harmonic behavior is due to the onset of methyl group rotations which exhibit a broad distribution of activated processes ( E a,ave = 12.2 kJ.mol (-1), sigma = 5.0 kJ x mol (-1)). Our results are likened to those reported for other proteins.