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The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V1 and VO sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28±2 kDa, indicating that this protein is dimeric. With a radius of gyration (Rg) and a maximum size (Dmax) of 2.7±0.2 nm and 8.0±0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G38–144 form (the carboxyl-terminus) was expressed and purified. G38–144 is homogeneous, with a molecular mass of approximately 12±3 kDa, indicating a monomeric form in solution. [Copyright &y& Elsevier]

Examines the biochemical and structural characterization of copper metallothionein in Saccharomyces cerevisiae. Determination of copper binding stoichiometry; Characterization of purified protein by electrophoresis; Role of metallothioneins in cellular defense.

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gramnegative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (> 90%) at different temperatures (20, 37, and 55 °C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20°C, i.e. below the β↔α acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 °C), 4.60 nm for Re lipopolysaccharide (at 37 °C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37°C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20°C. At higher concentrations of divalent cations (e. g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1:√2, 1:√3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration. [ABSTRACT FROM AUTHOR]