Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability

A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge–charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme.     

Conformational studies of heterochiral peptides with diastereoisomeric residues: Crystal and molecular structures of linear dipeptides derived from leucine,isoleucine, and allo-isoleucine

The x-ray diffraction analyses of three N- and C-terminally blocked L , D dipeptides, namely t-Boc-D -Leu-L -Leu-OMe ( 1 ), t-Boc-L -Ile-D -alle-OMe ( 2 ), and t-Boc-D -aIle-L -Ile-OMe (3) containing enantiomeric or diastereomeric amino acid residues have been carried out. The structures were determined by direct methods and refined anisotropically to final R factors of 0.077. 0.058. and 0.072 for ( 1 ) ( 2 ) and ( 3 ), respectively. Peptides 1–3 all assume a similar U-shaped structure with ? and ψ torsion angles cosrresponding to one of the possible calculated minimum energy regions (regions E and G for L residues, and F*. D* and H* for D residues). The peptide backbones of 1-3 are almost super-imposable [provided that the appropriate inversion of the chiral centers of ( 2 ) is made]. Side-chain conformations of Leu residues in peptide ( 1 ) are g^? (tg^?) for the L -Leu residue and the mirrored g⁺ (tg⁺) for the D -Leu residue; however, in peptides ( 2 ) and ( 3 ) the conformations of the isoconfiguralional side chains of the Ile or allo-Ile residues are (g^?t) t and (tg⁺) tfor the L -Ile and the D -allo-Ile moieties, respectively. In all cases, these conformations correspond to the more populated conformers of β-branched residues statistically found in crystal structures of small peptides. The results seem to indicate that, at least in short peptides with enantiomeric or diastereoisomeric residues, the change in chirality in the main-chain atoms perturbs the backbone conformation to a lesser extent and the side chain conformation to a greater extent. © 1995 John Wiley & Sons, Inc.     

Bioactive peptides: Conformational studies of [TYR4] cyclolinopeptide A

The solid state conformational analysis of [Tyr⁴] cyclolinopeptide A has been carried out by x-ray diffraction studies. The crystal structure of the monoclinic form, grown from a dioxane-water mixture [a = 9.849 (5) Å, b = 20.752 (4) Å, c = 16.728 (5) Å, β = 98.83 (3)°, space group P2₁, Z = 2], shows the presence of five intramolecular N-H? O?C hydrogen bonds, with formation of one C₁₇ ring structure, one α-turn (C₁₃), one inverse γ-turn (C₇), and two β-turns (C₁₀, one of type III and one of type 1). The Pro¹-Pro² peptide unit is cis (ω = 5°) all others are trans. The structure is almost superimposable with that of cyclolinopeptide A. The rms deviation for the atoms of the backbones is on the average 0.33 Å. © 1995 John Wiley & Sons, Inc.     

Effects of water deficit on the activity of nitrate reductase and content of sugars, nitrate and free amino acids in the leaves and roots of sunflower and white lupin plants growing under two nutrient supply regimes

The effects of soil drying on the activity of nitrate reductase (NR; EC 1.6.6.6) were studied in Helianthus annuus L. and non-nodulated Lupinus albus L. plants growing under two nutrient supply regimes. NR activity was assessed in leaf and root extracts by measuring the activity of the unphosphorylated active form (NR_act), the maximal extractable activity (NR_max) and the activation state. To obtain an insight into potential signalling compounds, nitrate, free amino acids and soluble sugars were also quantified. In both species, foliar NR_act and NR_max were negatively affected by soil drying and a decreased supply of nutrients, the observed changes in NR activity being linearly correlated with the depletion of nitrate. Similar results were obtained in the roots of sunflower. Conversely, in white lupin roots, NR_max was found to be independent of tissue nitrate concentration. Regardless of the species and organ, the activation state of the enzyme was unaffected by the nutrient supply regime. In well-watered sunflower roots, only about 50% of the existing NR was unphosphorylated, but the activation state increased significantly in response to drought. In contrast, lupin roots always exhibited NR activation state values close to 80%, or even higher. At the leaf level, the NR activation state was hardly changed in response to soil drying. The observed changes in the concentrations of soluble sugars and free amino acids are discussed in terms of their possible contribution to the variations in NR activity.     

Large-scale biofilm cultivation of Antarctic bacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC125 for physiologic studies and drug discovery

Microbial biofilms are mainly studied due to detrimental effects on human health but they are also well established in industrial biotechnology for the production of chemicals. Moreover, biofilm can be considered as a source of novel drugs since the conditions prevailing within biofilm can allow the production of specific metabolites. Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 when grown in biofilm condition produces an anti-biofilm molecule able to inhibit the biofilm of the opportunistic pathogen Staphylococcus epidermidis. In this paper we set up a P. haloplanktis TAC125 biofilm cultivation methodology in automatic bioreactor. The biofilm cultivation was designated to obtain two goals: (1) the scale up of cell-free supernatant production in an amount necessary for the anti-biofilm molecule/s purification; (2) the recovery of P. haloplanktis TAC125 cells grown in biofilm for physiological studies. We set up a fluidized-bed reactor fermentation in which floating polystyrene supports were homogeneously mixed, exposing an optimal air–liquid interface to let bacterium biofilm formation. The proposed methodology allowed a large-scale production of anti-biofilm molecule and paved the way to study differences between P. haloplanktis TAC125 cells grown in biofilm and in planktonic conditions. In particular, the modifications occurring in the lipopolysaccharide of cells grown in biofilm were investigated.     

Adaptive differentiation in floral traits in the presence of high gene flow in scarlet gilia (Ipomopsis aggregata)

Plant–pollinator interactions are thought to be major drivers of floral trait diversity. However, the relative importance of divergent pollinator‐mediated selection vs. neutral processes in floral character evolution has rarely been explored. We tested for adaptive floral trait evolution by comparing differentiation at neutral genetic loci to differentiation at quantitative floral traits in a putative Ipomopsis aggregata hybrid zone. Typical I. aggregata subsp. candida displays slender white tubular flowers that are typical of flowers pollinated by hawkmoths, and subsp. collina displays robust red tubular flowers typical of flowers pollinated by hummingbirds; yet, hybrid flower morphs are abundant across the East Slope of the Colorado Rockies. We estimated genetic differentiation (F_ST) for nuclear and chloroplast microsatellite loci and used a half‐sib design to calculate quantitative trait divergence (Q_ST) from collection sites across the morphological hybrid zone. We found little evidence for population structure and estimated mean F_ST to be 0.032. Q_ST values for several floral traits including corolla tube length and width, colour, and nectar volume were large and significantly greater than mean F_ST. We performed multivariate comparisons of neutral loci to genetic correlations within and between populations and found a strong signal for divergent selection, suggesting that specific combinations of floral display and reward traits may be the targets of selection. Our results show little support for historical subspecies categories, yet floral traits are more diverged than expected due to drift alone. Non‐neutral divergence for multivariate quantitative traits suggests that selection by pollinators is maintaining a correlation between display and reward traits.     

Modulation of membrane properties of lung cancer cells by azurin enhances the sensitivity to EGFR-targeted therapy and decreased β1 integrin-mediated adhesion

In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β₁ in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β₁ and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response