Browsing by Author "Jameson GB"
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- ItemDesign, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A(American Chemical Society, 2022-11-15) Kurup HM; Kvach MV; Harjes S; Barzak FM; Jameson GB; Harjes E; Filichev VVDrug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to cross-link two distant modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by the engineered C-terminal domain of A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3 inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine, a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar inhibitor of engineered A3BCTD and wild-type A3A (Ki = 690 ± 140 and 360 ± 120 nM, respectively), providing a platform for further development of powerful A3 inhibitors.
- ItemDigestive diversity and kinetic intrigue among heated and unheated β-lactoglobulin species.(ROYAL SOC CHEMISTRY, 2014-11) Loveday SM; Peram MR; Singh H; Ye A; Jameson GBFood processing often alters the structure of proteins, and proteins are deliberately denatured and aggregated to improve technological functionality in many cases. However, the digestive consequences of processing-induced alterations to protein structure have only recently been studied. Here we explored the process-structure-digestibility relationship in the context of heat-processing effects on the structure and gastric digestibility of the bovine whey protein β-lactoglobulin (β-lg). Heating β-lg produces an array of non-native monomers, dimers and aggregates, and we have characterised these with reverse-phase high performance liquid chromatography (RP-HPLC) as a complement to our earlier work using polyacrylamide gel electrophoresis (PAGE) techniques. Using a combination of SDS-PAGE and RP-HPLC we have identified pepsin-resistant dimers and peptides that appear early in digestion. In an unexpected finding, native β-lg underwent complete hydrolysis during prolonged incubation (48 h) with pepsin. Two phases of hydrolysis were identified, and the transition between phases appears to result from alterations to the secondary structure of β-lg at 3-4 h, as measured with circular dichroism spectroscopy, and/or the binding and release of a pepsin inhibitor peptide. This work has unpacked some of the complexities of the processing-structure-digestibility relationship in a highly simplified system; further work is needed to explore the implications of these findings for food processors, regulatory authorities and consumers.
- ItemManipulating and quantifying spin states in solution as a function of pressure and temperature(Royal Society of Chemistry, 2018-01-07) Hogue RW; Lepper CP; Jameson GB; Brooker SMonitoring the spin states of species in solution is a crucial aspect of understanding magnetic properties as well as spin-labile sensing, supramolecular, catalytic and biochemical processes. Herein, we describe the first quantitative variable-pressure and variable-temperature method of determining spin states in solution, demonstrate that it is accurate, and identify a simultaneous T and P sensor system.
- ItemModeling multiple duplex DNA attachments in a force-extension experiment(Cell Press, 2022-03-09) Raudsepp A; Williams MAK; Jameson GB; Enderlein JOptical tweezers-based DNA stretching often relies on tethering a single end-activated DNA molecule between optically manipulated end-binding beads. Measurement success can depend on DNA concentration. At lower DNA concentrations tethering is less common, and many trials may be required to observe a single-molecule stretch. At higher DNA concentrations tethering is more common; however, the resulting force-extensions observed are more complex and may vary from measurement to measurement. Typically these more complex results are attributed to the formation of multiple tethers between the beads; however, to date there does not appear to have been a critical examination of this hypothesis or the potential usefulness of such data. Here we examine stretches at a higher DNA concentration and use analysis and simulation to show how the more complex force-extensions observed can be understood in terms of multiple DNA attachments.
- ItemProgress toward Plug-and-Play Polymer Strings for Optical Tweezers Experiments: Concatenation of DNA Using Streptavidin Linkers(American Chemical Society, 2022-02-22) Mohandas N; Kent LM; Raudsepp A; Jameson GB; Williams MAKStreptavidin is a tetrameric protein that is renowned for its strong binding to biotin. The robustness and strength of this noncovalent coupling has led to multitudinous applications of the pairing. Within the streptavidin tetramer, each protein monomer has the potential to specifically bind one biotin-bearing moiety. Herein, by separating various streptavidin species that have had differing numbers of their four potential binding sites blocked, several different types of "linking hub" were obtained, each with a different valency. The identification of these species and the study of the plugging process used to block sites during their preparation were carried out using capillary electrophoresis. Subsequently, a specific species, namely, a trans-divalent linker, in which the two open biotin-binding pockets are approximately opposite one another, was used to concatenate two ∼5 kb pieces of biotin-terminated double-stranded DNA. Following the incubation of this DNA with the prepared linker, a fraction of ∼10 kb strings was identified using gel electrophoresis. Finally, these concatenated DNA strings were stretched in an optical tweezer experiment, demonstrating the potential of the methodology for coupling and extending molecules for use in single-molecule biophysical experiments.
- ItemSmall-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor(MDPI (Basel, Switzerland), 2021-02-12) Barzak FM; Ryan TM; Kvach MV; Kurup HM; Aihara H; Harris RS; Filichev VV; Harjes E; Jameson GB; Chelico LIn normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.
- ItemStructural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass(Portland Press on behalf of the Biochemical Society, 2018-10) Sterritt OW; Lang EJM; Kessans SA; Ryan TM; Demeler B; Jameson GB; Parker EJIn Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.
- ItemSynthesis and Characterization of Symmetrically versus Unsymmetrically Proton-Bridged Hexa-Iron Clusters(American Chemical Society, 2021-06-29) De Silva DNT; Dais TN; Jameson GB; Cutler DJ; Brechin EK; Davies CG; Jameson GNL; Plieger PGSyntheses and magnetic and structural characterization of hexa-iron complexes of derivatized salicylaldoximes are discussed. Complexation of Fe(BF4)2·6H2O with each ligand (H2 L1 and H4 L2) in a methanolic-pyridine solution resulted in hexa-iron compounds (C1 and C2, respectively), which each contain two near-parallel metal triangles of [Fe3-μ3-O], linked by six fluoride bridges and stabilized by a hydrogen-bonded proton between the μ3-O groups. Within each metal triangle of C2, Fe(III) ions are connected via the amine "straps" of (H4 L2-2H). Variable-temperature magnetic susceptibility and Mössbauer data of C1 and C2 indicate the presence of dominant antiferromagnetic interactions between the high-spin (S = 5/2) Fe(III) centers. For C1, two quadrupole doublets are observed at room temperature and 5 K, consistent with structural data from which discrete but disordered [Fe3-μ3-O] and [Fe3-μ3-OH] species were inferred. For C2, a single sharp quadrupole doublet with splitting intermediate between those determined for C1 was observed, consistent with the symmetric [Fe3-μ3-O···H···μ3-O-Fe3] species inferred crystallographically from the very short μ3-O···μ3-O separation. The differences in the physical properties of the complexes, as seen in the Mössbauer, X-ray, and magnetic data, are attributed to the conformational flexibility imparted by the nature of the linkages between the closely related ligands.
- ItemThe Effect of pH and Sodium Caseinate on the Aqueous Solubility, Stability, and Crystallinity of Rutin towards Concentrated Colloidally Stable Particles for the Incorporation into Functional Foods(MDPI (Basel, Switzerland), 2022-01-14) Rashidinejad A; Jameson GB; Singh H; Papetti APoor water solubility and low bioavailability of hydrophobic flavonoids such as rutin remain as substantial challenges to their oral delivery via functional foods. In this study, the effect of pH and the addition of a protein (sodium caseinate; NaCas) on the aqueous solubility and stability of rutin was studied, from which an efficient delivery system for the incorporation of rutin into functional food products was developed. The aqueous solubility, chemical stability, crystallinity, and morphology of rutin (0.1-5% w/v) under various pH (1-11) and protein concentrations (0.2-8% w/v) were studied. To manufacture the concentrated colloidally stable rutin-NaCas particles, rutin was dissolved and deprotonated in a NaCas solution at alkaline pH before its subsequent neutralisation at pH 7. The excess water was removed using ultrafiltration to improve the loading capacity. Rutin showed the highest solubility at pH 11, while the addition of NaCas resulted in the improvement of both solubility and chemical stability. Critically, to achieve particles with colloidal stability, the NaCas:rutin ratio (w/w) had to be greater than 2.5 and 40 respectively for the lowest (0.2% w/v) and highest (4 to 8% w/v) concentrations of NaCas. The rutin-NaCas particles in the concentrated formulations were physically stable, with a size in the range of 185 to 230 nm and zeta potential of -36.8 to -38.1 mV, depending on the NaCas:rutin ratio. Encapsulation efficiency and loading capacity of rutin in different systems were 76% to 83% and 2% to 22%, respectively. The concentrated formulation containing 5% w/v NaCas and 2% w/v rutin was chosen as the most efficient delivery system due to the ideal protein:flavonoid ratio (2.5:1), which resulted in the highest loading capacity (22%). Taken together, the findings show that the delivery system developed in this study can be a promising method for the incorporation of a high concentration of hydrophobic flavonoids such as rutin into functional foods.