Biomedical subjects

Olwyn Byron

Publications and source records attributed to Olwyn Byron.

12 recordsLinked to original sources

PubMed2006

A new level of architectural complexity in the human pyruvate dehydrogenase complex.

Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is a key metabolic assembly comprising a 60-meric pentagonal dodecahedral E2 (dihydrolipoamide acetyltransferase) core attached to which are 30 pyruvate decarboxylase E1 heterotetramers and 6 dihydrolipoamide dehydrogenase E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organization in which E3 and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 "cross-bridges" linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle x-ray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organization, its catalytic mechanism and regulation by the intrinsic PDC kinase.

Animals↗

PubMed2005

Behavior of Tn3 resolvase in solution and its interaction with res.

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.

Anisotropy↗

PubMed2005

SOMO (SOlution MOdeler) differences between X-Ray- and NMR-derived bead models suggest a role for side chain flexibility in protein hydrodynamics.

Reduced numbers of frictional/scattering centers are essential for tractable hydrodynamic and small-angle scattering data modeling. We present a method for generating medium-resolution models from the atomic coordinates of proteins, basically by using two nonoverlapping spheres of differing radii per residue. The computed rigid-body hydrodynamic parameters of BPTI, RNase A, and lysozyme models were compared with a large database of critically assessed experimental values. Overall, very good results were obtained, but significant discrepancies between X-ray- and NMR-derived models were found. Interestingly, they could be accounted for by properly considering the extent to which highly mobile surface side chains differently affect translational/rotational properties. Models of larger structures, such as fibrinogen fragment D and citrate synthase, also produced consistent results. Foremost among this method's potential applications is the overall conformation and dynamics of modular/multidomain proteins and of supramolecular complexes. The possibility of merging data from high- and low-resolution structures greatly expands its scope.

Algorithms↗

PubMed2004

Solution structure of the Tn3 resolvase-crossover site synaptic complex.

Tn3 resolvase is a site-specific DNA recombinase, which catalyzes strand exchange in a synaptic complex containing twelve resolvase subunits and two res sites. Hyperactive mutants of resolvase can form a simpler complex (X synapse) containing a resolvase tetramer and two shorter DNA segments at which strand exchange takes place (site I). We have solved the low-resolution solution structure of the purified, catalytically competent X synapse from small-angle neutron and X-ray scattering data, using methods in which the data are fitted with models constructed by rigid body transformations of a published crystallographic structure of a resolvase dimer bound to site I. Our analysis reveals that the two site I fragments are on the outside of a resolvase tetramer core and provides some information on the quaternary structure of the tetramer. We discuss implications of our structure for the architecture of the natural synaptic complex and the mechanism of strand exchange.

Bacterial Proteins↗

PubMed2004

Hydrodynamic bead modelling of the 2:1 p50-IkappaBgamma complex.

NFkappaB is an important and ubiquitous transcription factor formed by various homo- and heterodimers of the NFkappaB family. The active transcription factor regulates genes involved in immune, inflammatory and survival responses. Specificity in gene regulation is achieved, at least in part, by the distinct DNA binding preferences of the various homo- and heterodimers and by the complex pathways that lead to signal-induced degradation of the IkappaB inhibitors. Analytical ultracentrifugation and hydrodynamic bead modelling were used to model the solution structures of the NFkappaB family member p50, its inhibitor IkappaBgamma and their complex. Sedimentation equilibrium (SE) and sedimentation velocity (SV) data show that p50 is a dimer in solution with a sedimentation coefficient consistent with a conformation intermediate between the closed conformation observed in the crystal structure of the p50 (N-terminal domain)-p65 heterodimer complexed with IkappaBalpha and the open conformation adopted by p50 when bound to DNA. SE and SV data show that IkappaBgamma is a monomer in solution and is prone to aggregation over time. p50 forms a 2:1 stoichiometric complex with IkappaBgamma in solution with a sedimentation coefficient consistent with a closed conformation for the p50 dimer.

Attention↗

PubMed2004

Low-resolution reconstruction of a synthetic DNA holliday junction.

We have studied the low-resolution solution conformation of a Holliday (or four-way) DNA junction by using small-angle x-ray scattering, sedimentation velocity, and computational modeling techniques. The scattering data were analyzed in two independent ways: firstly, by rigid-body modeling of the scattering data using previously suggested models for the Holliday junction (HJ), and secondly, by ab initio reconstruction methods. The models found by both methods agree with experimentally determined sedimentation coefficients and are compatible with the results of previous studies using different techniques, but provide a more direct and accurate determination of the solution conformation of the HJ. Our results show that addition of Mg(2+) alters the conformation of the HJ from an extended to a stacked arrangement.

Algorithms↗

PubMed2004

The role of cholesterol in the activity of pneumolysin, a bacterial protein toxin.

The mechanism via which pneumolysin (PLY), a toxin and major virulence factor of the bacterium Streptococcus pneumoniae, binds to its putative receptor, cholesterol, is still poorly understood. We present results from a series of biophysical studies that shed light on the interaction of PLY with cholesterol in solution and in lipid bilayers. PLY lyses cells whose walls contain cholesterol. Using standard hemolytic assays we have demonstrated that the hemolytic activity of PLY is inhibited by cholesterol, partially by ergosterol but not by lanosterol and that the functional stoichiometry of the cholesterol-PLY complex is 1:1. Tryptophan (Trp) fluorescence data recorded during PLY-cholesterol titration studies confirm this ratio, reveal a significant blue shift in the Trp fluorescence peak with increasing cholesterol concentrations indicative of increasing nonpolarity in the Trp environment, consistent with cholesterol binding by the tryptophans, and provide a measure of the affinity of cholesterol binding: K(d) = 400 +/- 100 nM. Finally, we have performed specular neutron reflectivity studies to observe the effect of PLY upon lipid bilayer structure.

Amino Acid Motifs↗

PubMed2004

The solution structure and oligomerization behavior of two bacterial toxins: pneumolysin and perfringolysin O.

Pneumolysin (PLY), an important protein virulence factor of the human bacterial pathogen Streptococcus pneumoniae, could be a candidate for inclusion in a new anti-streptococcal vaccine. PLY solution species from monomer via multimeric intermediates to ring-shaped oligomers were studied with time-dependent sedimentation velocity in the analytical ultracentrifuge (AUC). Hydrodynamic bead modeling was used to interpret the data obtained. PLY remained mostly monomeric in solution; intermediate PLY multimers were detected in small quantities. Current understanding of PLY molecular mechanism is guided by a model built on the basis of its homology with perfringolysin O (PFO) for which there is an atomic structure. PFO, a virulence factor of the organism Clostridium perfringens, has almost the same molecular mass as PLY and shares 48% sequence identity and 60% sequence similarity with PLY. We report a comparative low-resolution structural study of PLY and PFO using AUC and small-angle x-ray scattering (SAXS). AUC data demonstrate that both proteins in solution are mostly monodisperse but PLY is a monomer whereas PFO is mostly dimeric. Ab initio dummy atom and dummy residue models for PFO and PLY were restored from the distance distribution function derived from experimental small-angle x-ray scattering curves. In solution, PLY is elongated, consistent with the shape predicted by its high-resolution homology model. The PFO dimer is also an elongated particle whose shape and volume are consistent with a staggered antiparallel dimer.

Bacterial Proteins↗

PubMed2003

Novel configurations of high molecular weight species of the pertussis toxin vaccine component.

Pertussis toxin (PT) is used in its formaldehyde-detoxified form in acellular pertussis vaccines for preventing whooping cough in children. The effects of formaldehyde treatment (up to 0.5% (w/v) formaldehyde) on the size, molecular association, folding and monoclonal antibody (mAb) binding of PT were studied to further define the structural nature of the high molecular weight species as related to their epitope integrity. Analytical ultracentrifugation (AUC) demonstrated that formaldehyde treatment of PT prevented the dissociation of the holotoxin. Together with results from size exclusion chromatography (SEC), SEC/multi-angle laser light scattering (MALLS) and immunoblotting it was demonstrated that PT increased in molecular weight and heterogeneity as a function of formaldehyde concentration, caused at least in part by covalent cross-linking. Five mAbs specific for PT subunits (S1-S5) bound to the cross-linked species, although there was some loss of epitopes in the larger aggregates. Intrinsic fluorescence spectroscopy gave evidence of progressive unfolding and re-association of PT. These findings demonstrate that a favourable balance between protein stabilisation and denaturation may be achieved by the treatment of pertussis toxin with formaldehyde, and provides a basis for determining the significance of high molecular weight cross-linked species of pertussis toxin in protection against whooping cough.

Amino Acid Substitution↗

PubMed2003

The polyprotein and FAR lipid binding proteins of nematodes: shape and monomer/dimer states in ligand-free and bound forms.

Nematodes produce two classes of small, helix-rich fatty acid- and retinol-binding proteins whose structures and in vivo functions remain to be elucidated. These are the polyprotein allergens (NPA) and the FAR proteins. The solution properties of recombinant forms of these proteins from parasitic [Ascaris suum (As) and Onchocerca volvulus (Ov)] and free-living [Caenorhabditis elegans (Ce)] nematodes have been examined. Analytical ultracentrifugation (AUC) showed that, contrary to previous findings, the rAs-NPA-1A polyprotein unit (approximately 15 kDa) is a monomer, and this stoichiometry is unaltered by ligand (oleic acid) binding. The rOv-FAR-1 and rCe-FAR-5 proteins differ in that the former forms a tight dimer and the latter a monomer, and these oligomeric states are also unaffected by ligand binding or protein concentration. Sedimentation equilibrium experiments showed that the partial specific volume v of the unliganded proteins agree well with the value calculated from amino acid composition extrapolated to experimental temperature, and was unaffected upon ligand binding. Data from small-angle X-ray scattering (SAXS) indicated that both of the monomeric proteins rAs-NPA-1A and rCe-FAR-5 are globular, although slightly elongated and flattened. These data are in good agreement with shapes predicted from sedimentation velocity experiments and hydrodynamic bead modelling. On the basis of functional and secondary structural homology with the ligand-binding domain of the retinoic acid receptor RXRalpha, de novo atomic resolution structures for rAs-NPA-1A and rCe-FAR-5 have been constructed which are consistent with the SAXS and hydrodynamic data.

Allergens↗

PubMed2003

19 A solution structure of the filarial nematode immunomodulatory protein, ES-62.

ES-62, a protein secreted by filarial nematodes, parasites of vertebrates including humans, has an unusual posttranslational covalent addition of phosphorylcholine to an N-type glycan. Studies on ES-62 from the rodent parasite Acanthocheilonema viteae ascribe it a dominant role in ensuring parasite survival by modulating the host immune system. Understanding this immunomodulation at the molecular level awaits full elucidation but distinct components of ES-62 may participate: the protein contributes aminopeptidase-like activity whereas the phosphorylcholine is thought to act as a signal transducer. We have used biophysical and bioinformatics-based structure prediction methods to define a low-resolution model of ES-62. Sedimentation equilibrium showed that ES-62 is a tightly bound tetramer. The sedimentation coefficient is consistent with this oligomer and the overall molecular shape revealed by small angle x-ray scattering. A 19 A model for ES-62 was restored from the small-angle x-ray scattering data using the program DAMMIN which uses simulated annealing to find a configuration of densely packed scattering elements consistent with the experimental scattering curve. Analysis of the primary sequence with the position-specific iterated basic local alignment search tool, PSI-BLAST, identified six closely homologous proteins, five of which are peptidases, consistent with observed aminopeptidase activity in ES-62. Differences between the secondary structure content of ES-62 predicted using the consensus output from the secondary structure prediction server JPRED and measured using circular dichroism are discussed in relation to multimeric glycosylated proteins. This study represents the first attempt to understand the multifunctional properties of this important parasite-derived molecule by studying its structure.

Amino Acid Sequence↗

PubMed2002

Low resolution solution structure of the Apo form of Escherichia coli haemoglobin protease Hbp.

We have studied the solution properties of the apo form of the haemoglobin protease or "haemoglobinase", Hbp, a principal component of an important iron acquisition system in pathogenic Escherichia coli. Experimental determination of secondary structure content from circular dichroism (CD) spectroscopy, obtained using synchrotron light, showed that the protein contains predominately beta-sheets in agreement with secondary structure prediction from the primary sequence. Next, the size and shape of the protein were probed using analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). These showed that Hbp is a monomer, with an extended conformation. Using ab initio reconstruction methods we have produced a model of Hbp, which shows that the protein adopts an extended crescent-shaped conformation. Analysis of the resulting model gives hydrodynamic parameters in good agreement with those observed experimentally. Thus we are able to construct a hydrodynamically rigorous model of apo-Hbp in solution, not only giving a greater level of confidence to the results of the SAXS reconstruction methods, but providing the first three-dimensional view of this intriguing molecule.

Apoenzymes↗