Browsing by Author "Jameson, GB"

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    Evolution of quaternary structure in a homotetrameric enzyme
    (Elsevier, 2008-07-18) Griffin, MDW; Dobson, RCJ; Pearce, FG; Antonio, L; Whitten, AE; Liew, K; Mackay, JP; Trewhella, J; Jameson, GB; Perugini, MA; Gerrard, JA
    Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits. © 2008, Elsevier Ltd.
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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