Browsing by Author "Ryan, TM"

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    Combined pressure and temperature denaturation of ribonuclease A produces alternate denatured states
    (Elsevier, 2016-05-13) Ryan, TM; Xun, Y; Cowieson, NP; Mata, JP; Jackson, AJ; Pauw, BR; Smith, AJ; Kirby, N; McGillivray, DJ
    Protein folding, unfolding and misfolding have become critically important to a range of health and industry applications. Increasing high temperature and high pressure are used to control and speed up reactions. A number of studies have indicated that these parameters can have a large effect on protein structure and function. Here we describe the additive effects of these parameters on the small angle scattering behaviour of ribonuclease A. We find that alternate unfolded structures can be obtained with combined high pressure and temperature treatment of the protein. © 2016 Elsevier Inc.
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    Domain organization of the monomeric form of the Tom70 mitochondrial import receptor
    (Elsevier, 2009-05-22) Mills, RD; Trewhella, J; Qiu, TW; Welte, T; Ryan, TM; Hanley, TL; Knott, RB; Lithgow, T; Mulhern, TD
    Tom70 is a mitochondrial protein import receptor composed of 11 tetratricopeptide repeats (TPRs). The first three TPRs form an N-terminal domain that recruits heat shock protein family chaperones, while the eight C-terminal TPRs form a domain that receives, from the bound chaperone, mitochondrial precursor proteins destined for import. Analytical Ultracentrifugation and solution small-angle X-ray scattering (SAXS) analysis characterized Tom70 as an elongated monomer. A model for the Tom70 monomer was proposed based on the alternate interpretation of the domain pairings observed in the crystal structure of the Tom70 dimer and refined against the SAXS data. In this "open" model of the Tom70 monomer, the chaperone- and precursor-binding sites are exposed and lay side by side oil one face of the molecule. Fluorescence anisotropy measurements indicated that monomeric Tom70 can bind both chaperone and precursor peptides and that chaperone peptide binding does not alter the affinity of Tom70 for the precursor peptide. SAXS Was unable to detect any shape change in Tom70 upon chaperone binding. However, molecular modeling indicated that chaperone binding is incompatible with Tom70 dimer formation. It is proposed that the Tom70 monomer is the functional unit mediating initial chaperone docking and precursor recognition. © 2009, Elsevier Ltd.
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    Grouper iridovirus GIV66 is a Bcl-2 protein that inhibits apoptosis by exclusively sequestering Bim
    (Society of Crystallographers in Australia and New Zealand, 2017-12-03) Banjara, S; Mao, J; Ryan, TM; Caria, S; Kvansakul, M
    Programmed cell death or apoptosis is a critical mechanism for the controlled removal of damaged or infected cells, and proteins of the Bcl-2 family are important arbiters of this process. Viruses have been shown to encode for functional and structural homologues of Bcl-2 to counter premature host cell apoptosis to ensure viral proliferation and/or survival. Grouper iridovirus (GIV) is a large DNA virus belonging to the iridoviridae family that harbours GIV66, a putative Bcl-2 like protein. GIV66 is a mitochondrially localised inhibitor of apoptosis, however, the molecular and structural basis of apoptosis inhibition is currently not understood. To gain insight into the mechanism of action we systematically evaluated the ability of GIV66 to bind peptides spanning the BH3 domain of proapoptotic Bcl-2 family members. Our data reveal that GIV66 harbours an unusually high level of specificity for pro-apoptotic Bcl-2, and only engages with Bim. We then determined crystal structures of both GIV66 on its own as well as bound to Bim BH3. Unexpectedly, GIV66 forms dimers via a novel interface that occludes access to the canonical Bcl-2 ligand binding groove, which break apart upon Bim binding. These data suggest that GIV66 dimerisation impacts on the ability of GIV66 to bind and select host pro-death Bcl-2 protein. Our findings provide a mechanistic understanding for the potent anti-apoptotic activity of GIV66 by identifying it as the first single specificity pro-survival Bcl2 protein, and demonstrating the pivotal role of Bim for GIV mediated inhibition of apoptosis.
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    Mechanistic understanding of PHD2 enzyme upon ligand interactions using SAXS and SANS
    (International Conference on Neutron Scattering, 2017-07-12) Vadakkedath, PG; Ryan, TM; Leung, IKH; McGillivray, DJ
    Prolyl hydroxylase domain proteins (PHDs) play an important role in the regulation of cellular homeostasis in response to changes in cellular oxygen level.1 In the presence of oxygen, PHDs catalyse the oxygen-dependent hydroxylation of hypoxia inducible factor (HIF), which lead to its degradation. However, in the absence of oxygen, HIF cannot be hydroxylated and can therefore trigger downstream hypoxic responses including the formation of new blood vessels and red bloodcells. PHDs catalyse the hydroxylation of HIF at two different proline residues.2–3 We are interested in understanding the structural basis in the substrate selectivity of PHDs. Using small angle X-ray scattering (SAXS), we examined the conformational changes of PHD2 particularly ?2 ?3 loop in the presence of CODD and NODD substrates where we see change in overall size and shape of PHD2. We obtained PHD2-NODDsolution structure as a confirmation to crystallographic structure3. We would further confirm these conformational changes upon ligand interactions using Small angle neutron scattering. It would help us in understand ?2 ?3 loopand ?4 regions of PHD2 which is crucial in binding of CODD and NODD substrates. The contribution of ?2 ?3 loop and ?4 regions of PHD2 in protecting the active site is known from the NMR data2–3. Both SAXS and SANS would be complementary to NMR data in solution which would further help us in design PHD2 inhibitors for hypoxia related diseases like ischemic heart disease and anaemia.
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    Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation
    (Royal Society of Chemistry, 2017-03-14) Xu, AY; Melton, LD; Ryan, TM; Mata, JP; Jameson, GB; Rekas, A; Williams, MAK; McGillivray, DJ
    We have simplified the structural heterogeneity of protein–polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (βLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the βLgA–OGA interaction is critically dependent on the length of the oligosaccharide. Isothermal titration calorimetry results suggest that a minimum length of 7 or 8 sugar residues is required in order to exhibit appreciable exothermic interactions with βLgA – shorter oligosaccharides show no enthalpic interactions at any concentration ratio. When titrating βLgA into OGAs with more than 7–8 sugar residues the sample solution also became turbid with increasing amounts of βLgA, indicating the formation of macroscopic assemblies. Circular dichroism, thioflavin T fluorescence and small angle X-ray/neutron scattering experiments revealed two structural regimes during the titration. When OGAs were in excess, βLgA formed discrete assemblies upon OGA binding, and no subsequent aggregation was observed. However, when βLgA was present in excess, multi-scale structures were formed and this eventually led to the separation of the solution into two liquid-phases. © 2017 The Royal Society of Chemistry