Aggregatibacter Actinomycetemcomitans Leukotoxin Cytotoxicity Occurs Through Bilayer Destabilization

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization. © 2012 Blackwell Publishing Ltd

Molecular structure, dynamics and hydration studies of soybean storage proteins and model systems by nuclear magnetic resonance

The potential of high-resolution \sp{13}C NMR for the characterization of soybean storage proteins was explored. The spectra of a commercial soy protein isolate as well as those of alkali-denatured 7S and 11S soybean globulins were well resolved and tentatively assigned. Relaxation measurements indicated fast motion for several side chains and the protein backbone. Protein fractions (11S and 7S) were also investigated at various states of molecular association. The large size of the multisubunit soybean storage proteins affected adversely both the resolution and the sensitivity of their \sp{13}C NMR spectra. A comparison of \sp{17}O and \sp2H NMR relaxation rates of water in solutions of lysozyme (a model system) as a function of concentration, pH and magnetic field suggested that only \sp{17}O monitors directly the hydration of lysozyme. Analysis of \sp{17}O NMR lysozyme hydration data in terms of a two-state, fast-exchange, anisotropic model resulted in hydration parameters which are consistent with the protein's physico-chemical properties. The same model was applied to the calculation of the amount and mobility of 'bound' water in soy protein dispersions by means of \sp{17}O NMR relaxation measurements as a function of protein concentration. The protein concentration dependences of \sp1H transverse NMR relaxation measurements at various pH and ionic strength values were fitted by a virial expansion. The interpretation of the data was based on the effects of protein aggregation, salt binding and protein group ionization on the NMR measurements. In all cases, relaxation rates showed a linear dependence on protein activity.U of I OnlyETDs are only available to UIUC Users without author permissio

Determination of Pre-Steady-State Rate Constants on the Escherichia coli Pyruvate Dehydrogenase Complex Reveals That Loop Movement Controls the Rate-Limiting Step

Spectroscopic identification and characterization of covalent and noncovalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. The Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), consisting of multiple copies of enzymic components and coenzymes, performs the oxidative decarboxylation of pyruvate to acetyl-CoA and is central to carbon metabolism linking glycolysis to the Krebs cycle. On the basis of earlier studies, we hypothesized that the dynamic regions of the E1p component, which undergo a disorder–order transition upon substrate binding to thiamin diphosphate (ThDP), play a critical role in modulation of the catalytic cycle of PDHc. To test our hypothesis, we kinetically characterized ThDP-bound covalent intermediates on the E1p component, and the lipoamide-bound covalent intermediate on the E2p component in PDHc and in its variants with disrupted active-site loops. Our results suggest that formation of the first covalent predecarboxylation intermediate, C2α-lactylthiamin diphosphate (LThDP), is rate limiting for the series of steps culminating in acetyl-CoA formation. Substitutions in the active center loops produced variants with up to 900-fold lower rates of formation of the LThDP, demonstrating that these perturbations directly affected covalent catalysis. This rate was rescued by up to 5-fold upon assembly to PDHc of the E401K variant. The E1p loop dynamics control covalent catalysis with ThDP and are modulated by PDHc assembly, presumably by selection of catalytically competent loop conformations. This mechanism could be a general feature of 2-oxoacid dehydrogenase complexes because such interfacial dynamic regions are highly conserved

Elucidation of the Interaction Loci of the Human Pyruvate Dehydrogenase Complex E2·E3BP Core with Pyruvate Dehydrogenase Kinase 1 and Kinase 2 by H/D Exchange Mass Spectrometry and Nuclear Magnetic Resonance

The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1–4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S′ induced moderate interaction with both PDKs according to both methods