Luigi Leonardo Palese

Metaproteome analysis of sewage sludge from membrane bioreactors

Microbial dynamics and enzymatic activities of activated sludge processes are not completely understood yet. A better understanding about the biology is indispensable for further process optimization. Since proteins play a key role as catalysts in sludge processes, a protocol for protein extraction and analysis by 2‐D PAGE was established. It is based on phenol extraction of alkaline extracts and on a subsequent precipitation with ammonium sulphate. 2‐D protein patterns obtained from different sludges collected from membrane bioreactors showed – besides common spots – significant differences. Selected proteins were identified with nano‐HPLC‐ESI‐MS/MS. All membrane biological reactor (MBR) sludge samples investigated in this study contained elastase 3A, which implies that this human serine protease is a significant constituent of municipal wastewater. Although the identification of proteins from ammonia‐oxidizing bacterium Nitrosomonas europaea was expected, the detection of a protein with homology to the marine bacterium Saprospira grandis in MBR1 was surprising.

Design of mineral medium for growth of Actinomadura sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources.

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin, with significant activity against Neisseria gonorrhoeae and precursor of the semi-synthetic antibiotic dalbavancin. In this study the production of A40926 by Actinomadura under a variety of growth conditions was investigated. The use of chemically defined mineral media allowed us to analyze the influence of carbon and nitrogen sources, phosphate, ammonium and calcium on the growth and the antibiotic productivity of Actinomadura. We confirm recent data [Gunnarsson et al. (2003) J Ind Microbiol Biotechnol 30:150–156] that low initial concentrations of phosphate and ammonium are beneficial for growth and A40926 production, and we provide new evidence that the production of A40926 is depressed by calcium, but promoted when l-glutamine or l-asparagine are used as nitrogen sources instead of ammonium salts.

Cooperative coupling and role of heme a in the proton pump of heme-copper oxidases

In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanisms, to heme-copper oxidases. The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers. The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit I of the oxidase. Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation. Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts. Two groups result in being linked to redox transitions of heme a3. One group is apparently linked to CuB. The fourth group is linked to oxido-reduction of heme a. We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and CuB in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals. This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism. The group linked to heme a, which can transfer up to 0.9 H+/e- at pHs around neutrality, can provide the major contribution to the proton pump. It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center. The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conversion of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle. Site-directed mutagenesis of protolytic residues in subunit I of the aa3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase. Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate. Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.

Protein dynamics: Complex by itself

Biological functions are intimately rooted in biopolymer dynamics. It is commonly accepted that proteins can be considered as complex systems, but the origin of such complexity is still not fully understood. Moreover, it is still not really clear if proteins are true complex systems or complicated ones. Here, molecular dynamics simulations on a two helix bundle protein have been performed, and protein trajectories have been analyzed by using correlation functions in the frequency domain. We show that even a simple protein exhibits the hallmarks of complex systems. Moreover, the molecular bases of this complex behavior are possessed completely by the protein itself, because such complexity emerges without considering the solvent explicitly.

Amyloid beta(1–42) in aqueous environments: Effects of ionic strength and E22Q (Dutch) mutation

Development of extracellular plaques characteristic of Alzheimers disease is related to aggregation of amyloid peptides. The Aβ-42 peptide is the most aggregation prone species, and some missense mutant forms increase this aggregation ability. Due to its poor solubility as monomer in aqueous solutions, Aβ-42 conformational transitions in water have been largely investigated by molecular dynamics. Here we report an all-atom molecular dynamics analysis of the Aβ-42 peptide in aqueous environment using as starting conformation a structure obtained in an isotropic, low-polarity medium, representing a plausible model for the membrane-bound species. While previous studies commonly show that Aβ-42 is largely unstructured in aqueous solution, here we report that this peptide can adopt partially folded structures. Importance of ionic strength has been also investigated, showing that at physiological ionic strength condition a loop stabilizing electrostatic interaction involving Lys28 builds up. In addition, besides stable α-helix structures, we observe the appearance of 310 helix, similar to what was reported experimentally for the Aβ-40 species. The effect of E22Q (Dutch) mutation in high ionic strength condition has been explored. We show that this mutation has a dramatic impact on the Aβ-42 structure. Instead of a partially folded, but extended, conformation obtained with the wild type, the E22Q assumes a two-helix collapsed one due to the clustering of hydrophobic residues.

Molecular dynamics in cytochrome c oxidase Mössbauer spectra deconvolution

In this work low temperature molecular dynamics simulations of cytochrome c oxidase are used to predict an experimentally observable, namely Mössbauer spectra width. Predicted lineshapes are used to model Lorentzian doublets, with which published cytochrome c oxidase Mössbauer spectra were simulated. Molecular dynamics imposed constraints to spectral lineshapes permit to obtain useful information, like the presence of multiple chemical species in the binuclear center of cytochrome c oxidase. Moreover, a benchmark of quality for molecular dynamic simulations can be obtained. Despite the overwhelming importance of dynamics in electron-proton transfer systems, limited work has been devoted to unravel how much realistic are molecular dynamics simulations results. In this work, molecular dynamics based predictions are found to be in good agreement with published experimental spectra, showing that we can confidently rely on actual simulations. Molecular dynamics based deconvolution of Mössbauer spectra will lead to a renewed interest for application of this approach in bioenergetics.

Allosteric interactions and proton conducting pathways in proton pumping aa3 oxidases: Heme a as a key coupling element ☆

In this paper allosteric interactions in protonmotive heme aa(3) terminal oxidases of the respiratory chain are dealt with. The different lines of evidence supporting the key role of H(+)/e(-) coupling (redox Bohr effect) at the low spin heme a in the proton pump of the bovine oxidase are summarized. Results are presented showing that the I-R54M mutation in P. denitrificans aa(3) oxidase, which decreases by more than 200mV the E(m) of heme a, inhibits proton pumping. Mutational amino acid replacement in proton channels, at the negative (N) side of membrane-inserted prokaryotic aa(3) oxidases, as well as Zn(2+) binding at this site in the bovine oxidase, uncouples proton pumping. This effect appears to result from alteration of the structural/functional device, closer to the positive, opposite (P) surface, which separates pumped protons from those consumed in the reduction of O(2) to 2 H(2)O.

Characterization of plasma membrane respiratory chain and ATPase in the actinomycete Nonomuraea sp. ATCC 39727

We have characterized the respiratory system of the aerobic actinomycete Nonomuraea sp. ATCC 39727. The plasma membrane of the microorganism is shown to contain a protonmotive respiratory chain and H+-ATPase. The respiratory chain is made up of a rotenone-sensitive NADH-quinone oxidoreductase, a four subunits aa3-type cytochrome c oxidase and a bc1 complex. The H+-ATPase is characterized as an F0F1-type on the basis of its sensitivity to specific inhibitors; the enzyme is also inhibited by mM concentrations of Ca2+. The activity of the respiratory chain increases during the exponential growth phase, but is depressed in the stationary phase. The H+-ATPase activity reaches, as the respiratory chain, a maximal activity at the end of the exponential growth phase and then remains constant in the stationary phase.

Random Matrix Theory in molecular dynamics analysis.

It is well known that, in some situations, principal component analysis (PCA) carried out on molecular dynamics data results in the appearance of cosine-shaped low index projections. Because this is reminiscent of the results obtained by performing PCA on a multidimensional Brownian dynamics, it has been suggested that short-time protein dynamics is essentially nothing more than a noisy signal. Here we use Random Matrix Theory to analyze a series of short-time molecular dynamics experiments which are specifically designed to be simulations with high cosine content. We use as a model system the protein apoCox17, a mitochondrial copper chaperone. Spectral analysis on correlation matrices allows to easily differentiate random correlations, simply deriving from the finite length of the process, from non-random signals reflecting the intrinsic system properties. Our results clearly show that protein dynamics is not really Brownian also in presence of the cosine-shaped low index projections on principal axes.

Relationship between cardiolipin metabolism and oxygen availability in Bacillus subtilis

We report changes of the content of anionic phospholipids in Bacillus subtilis in response to hypoxic conditions and inhibition of terminal respiration. Cardiolipin accumulates rapidly when bacteria are suspended in non‐growth medium under reduced aeration or exposed to the inhibitor cyanide; the increase of cardiolipin occurs at the expense of its precursor phosphatidylglycerol and is temperature‐dependent. Depending on the extent of hypoxic stress, membranes containing different levels of cardiolipin can be isolated from B. subtilis cells. The NADH oxidase activity in cardiolipin‐enriched membranes is cyanide‐resistant; furthermore O2 consumption measurements indicated that cardiolipin‐enriched cells are resistant to cyanide. Results point out a possible interdependence between the effect of cyanide on cardiolipin metabolism and the effect of cardiolipin on the effectiveness of cyanide inhibition.