Molecular camouflage : making use of protecting groups to control the self-assembly of inorganic janus particles onto metal–chalcogenide nanotubes by Pearson Hardness

Hard and soft: Binding of inorganic Pt@Fe3O4 Janus particles to WS2 nanotubes through their Pt or Fe3O4 domains is governed by the difference in Pearson hardness: the soft Pt block has a higher sulfur affinity than the harder magnetite face; thus the binding proceeds preferentially through the Pt face. This binding preference can be reversed by masking the Pt face with an organic protecting group.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

A computer generated model of adenosine receptors rationalising binding and selectivity of receptor ligands

Computer graphic analyses on a broad spectrum of adenosine receptor ligands has shown that both the A1 and A2 adenosine receptors have three binding sites. The spatial relationship of these three binding sites has been defined. Adenosine orientation at A1 and A2 is different.

Microtubulin binding sites as target for developing anticancer agents

Microtubules (MTs) play important and diverse roles in eukaryotic cells. Their function and biophysical properties have made α−and β−tubulin, the main components of MTs, the subject of intense study. Interfering with normal MT dynamics, for example, by the addition of tubulin ligands, can cause the cell great distress and affect MT stability and functions, including mitosis, cell motion and intracellular organelle transport. It has been shown in the literature that tubulin is an important target molecule for developing anticancer drugs. Tubulin binding molecules have generated considerable interest after the successful introduction of the taxanes into clinical oncology and the widespread use of the vinca alkaloids vincristine and vinblastine. These compounds inhibit cell mitosis by binding to the protein tubulin in the mitotic spindle and preventing polymerization into the MTs. This mode of action is also shared with other natural agents eg colchicine and podophyllotoxin. However various tubulin isotypes have shown resistance to taxanes and other MT agents. Therefore, there is a strong need to design and develop new natural analogs as antimitotic agents to interact with tubulin at sites different from those of vinca alkaloids and taxanes. This minireview provides SAR on several classes of antimitotic agents reported in the literature. The structures and data given are essential to the scientists who are involved in drug design and development in the field of anticancer drugs.

The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a ‘simple’ domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these ‘simple’ SSBs interact with ssDNA.

Differential interaction between the C2B domain of synaptotagmin-I and the Drosophila stonedA and stonedB proteins

The stoned locus in Drosophila encodes two proteins StonedA (STNA) and StonedB (STNB), both of which have been suggested to act as adaptins in mediating synaptic vesicle recycling. A combination of immunological, genetic and biochemical studies have shown an interaction of STNA and STNB with the C2B domain of Synaptotagmin-I (SYT-1), an integral synaptic vesicle protein that mediates Ca2+-dependent exocytosis, as well as endocytosis. The C2B domain of SYT-1 contains an AP-2 binding site that controls the size of recycled vesicles, and a C-terminal tryptophan-containing motif that acts as an internalization signal. Investigation of SYT-1 mutations in Drosophila has shown that altering the Ca2+ binding region of the C2B domain, results in a reduction in the rate of vesicle recycling, implicating this region in SYT-I endocytosis. In this poster, we report the molecular dissection of the interactions between the STNA and STNB proteins and the C2B domain of SYT-1. Deletion of the AP-2 binding site decreased the binding of both STNA and STNB. However, C-terminal deletions of the C2B domain significantly increased STNB binding. In contrast, the same C-terminal deletions reduced the affinity of the C2B domain for STNA. The possible interactions of both STNB and STNA with the Ca2+ binding region of SYT-1 will be also investigated.

Service composition and binding

Service composition enables the creation of services previously unavailable through the aggregation of existing services. The result is called a service composition. Exposing a service composition as a service, the result is called a composed service. It can be distinguished from atomic services. Service composition approaches can be differentiated along two axes: point in time of composition and degree of automation. With design-time and run-time we can identify two different points in time for doing a composition. Additionally we can distinguish between three different degrees of automation: manual, assisted, and automated service composition. © 2008 Springer Berlin Heidelberg.

Investigation of the binding interactions between insulin and its receptor

This project investigated the interactions between insulin and its receptor. A combination of computational and experimental investigations resulted in the identification of four residues in non-canonical sites that, when mutated, had detrimental effects on insulin binding. An increased understanding of the binding mechanism will aid future research into diseases involving the insulin receptor and its relatives and could potentially lead to new therapeutic avenues to combat these health related issues.

Email negotiations form a binding land contract without a 'signature'

Email is rapidly replacing other forms of communication as the preferred means of communication between contracting parties. The recent decision of Stellard Pty Ltd v North Queensland Fuel Pty Ltd [2015] QSC 119 reinforces the judicial acceptance of email as an effective means of creating a binding agreement and the willingness to adopt a liberal concept of ‘signing’ in an electronic environment.