Permeability and photoxidative damage of membrane enzymes

The pattern of photodynamic damage of pig erythrocyte and rat brain microsomal ATPases and erythrocyte acetylcholinesterases has been studied, using rose bengal as photosensitizer. Of these enzymes the Na⁺-K⁺-Mg²⁺-ATPase, believed to be associated with active transport, is very much more sensitive to damage than are the Mg²⁺-ATPase and the erythrocyte acetylcholinesterase. Earlier photoxidative studies of the haemolysis of erythrocytes have shown that ion movements occur in two phases which are dependent on the duration of exposure to light and it is suggested that these are correlated with the differential sensitivity of these membrane enzymes. The ATPases of the brain microsomal preparation were more sensitive to photodynamic damage during preincubation at 0°C, i.e. in the absence of substrate. Raising the preincubation temperature to 37°C protected both enzymes, the inactivation of the Na⁺-K⁺-Mg²⁺-ATPase being markedly reduced. The presence of substrate during pre-incubation at 0°C also protects both enzymes, especially the Mg²⁺-ATPase. These interacting effects of temperature and substrate are compared with the known different temperature sensitivities of these two enzymes.     

Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants.

In plants, environmental adversity often leads to the formation of highly reactive oxygen radicals. Since resistance to such conditions may be correlated with the activity of enzymes involved in oxygen detoxification, we have generated transgenic tobacco plants which express elevated levels of manganese superoxide dismutase (MnSOD) within their chloroplasts or mitochondria. Leaf discs of these plants have been analyzed in conditions in which oxidative stress was generated preferentially within one or the other organelle. It was found that high level overproduction of MnSOD in the corresponding subcellular location could significantly reduce the amount of cellular damage which would normally occur. In contrast, small increases in MnSOD activity were deleterious under some conditions. A generally applicable model correlating the consequences of SOD with the magnitude of its expression is presented.     

The S. pombe Translation Initiation Factor eIF4G Is Sumoylated and Associates with the SUMO Protease Ulp2

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

Ground state structure of F1-ATPase from bovine heart mitochondria at 1.9 A resolution

The structure of bovine F(1)-ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9A resolution. This structure has been compared with the previously described structure of bovine F(1)-ATPase determined at 1.95A resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the beta(DP)-subunit. In the present structure, the nucleotide binding sites in the beta(DP)- and beta(TP)-subunits are both occupied by AMP-PNP, whereas in the earlier structure, the beta(TP) site was occupied by AMP-PNP and the beta(DP) site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, alphaArg-373 differs in the beta(DP)- and beta(TP)-subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.     

Electron-electron distances in spin-labeled low-spin metmyoglobin variants by relaxation enhancement

Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure resulted in a protein with fewer hemichrome impurities than was obtained by an overexpression of holo-protein followed by spin labeling. Coordination of cyanide to the met heme formed low-spin complexes. Iron-nitroxyl interspin distances in the range of 17-30 Å were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between ∼30-140 K, and by spin-echo measurements of the enhancement of spin-spin relaxation rates at 10-30 K. Interspin distances were also calculated, using the molecular modeling program Insight II (Accelrys, San Diego, CA). For most variants, distances determined from the temperature dependence of spin-echo intensities at a pulse spacing of 200 ns agree with distances measured by saturation recovery and calculated with Insight II within about an angstrom, which is within experimental uncertainties. Measurements of interspin distances via spin-spin relaxation enhancement have the advantages that maximum effects are observed for slower metal relaxation rates than are required for spin-lattice relaxation enhancement, and the impact diminishes as r⁻³ instead of r⁻⁶, as with spin-lattice relaxation enhancement, which permits measurements at longer distances.     

PiuA and PiaA, iron uptake lipoproteins of Streptococcus pneumoniae, elicit serotype independent antibody responses following human pneumococcal septicaemia

Streptococcus pneumoniae causes considerable morbidity and mortality worldwide. The need for a cheap and effective pneumococcal vaccine has necessitated the evaluation of common virulence-associated proteins as potential vaccine antigens. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters. Here, we show that patients with culture confirmed pneumococcal septicaemia have elevated levels of antibody to PiuA and PiaA in convalescent-phase, compared with acute-phase serum. Additionally, sera from septicaemic patients infected with 13 pneumococcal strains covering eight different serotypes, cross-reacted with recombinant PiuA-His(6) and PiaA-His(6) from a single pneumococcal strain, indicating that this immune response is serotype independent. Anti-PiuA and anti-PiaA antibodies were also found in healthy seven-month-old infants, indicating that they are immunogenic at a very early age.     

Thermal denaturation of iso-1-cytochrome c variants: comparison with solvent denaturation.

Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and Tm at various pH values, the heat capacity increment (delta Cp), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. For the Trp 73 variant, the delta Cp value (1.15 +/- 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 +/- 0.06 kcal/mol K), while for the Ile 73 (1.65 +/- 0.07 kcal/mol K) and the Val 73 (1.50 +/- 0.06 kcal/mol K) variants, delta Cp increased slightly. In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Hermann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues. Biochemistry 34:3040-3047). Both the m-value and delta Cp are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-1-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of m-values and delta Cp for these variants will be discussed in this context.