High Mitochondrial DNA Copy Number Is a Protective Factor From Vision Loss in Heteroplasmic Leber's Hereditary Optic Neuropathy (LHON).

PURPOSE: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease that typically causes bilateral blindness in young men. It is characterized by as yet undisclosed genetic and environmental factors affecting the incomplete penetrance. METHODS: We identified 27 LHON subjects who possess heteroplasmic primary LHON mutations. Mitochondrial DNA (mtDNA) copy number was evaluated. RESULTS: The presence of centrocecal scotoma, an edematous, hyperemic optic nerve head, and vascular tortuosity, as well as telangiectasia was recognized in affected subjects. We found higher cellular mtDNA content in peripheral blood cells of unaffected heteroplasmic mutation carriers with respect to the affected. CONCLUSIONS: The increase of cellular mtDNA content prevents complete loss of vision despite the presence of a heteroplasmic state of LHON primary mutation, suggesting that it is a key factor responsible for penetrance of LHON.

Protein States as Symmetry Transitions in the Correlation Matrices.

Over the last few years, there has been significant progress in the knowledge on protein folding. However, some aspects of protein folding still need further attention. One of these is the exact relationship between the folded and unfolded states and the differences between them. Whereas the folded state is well known, at least from a structural point of view (just think of the thousands of structures in online databases), the unfolded state is more elusive. Also, these are dynamic states of matter, and this aspect cannot be overlooked. Molecular dynamics-derived correlation matrices are an invaluable source of information on the protein dynamics. Here, bulk eigenvalue spectra of the correlation matrices obtained from the Trp-cage dynamics in the folded and unfolded states have been analyzed. The associated modes represent localized vibrations and are significantly affected by the fine details of the structure and interactions. Therefore, these bulk modes can be used as probes of the protein local dynamics in different states. The results of these analyses show that the correlation matrices describing the folded and unfolded dynamics belong to different symmetry classes. This finding provides new support to the phase-transition models of protein folding.

Correlation Analysis of Trp-Cage Dynamics in Folded and Unfolded States.

A fundamental and still debated problem is how folded structures of proteins are related to their unfolded state. Besides the classical view, in which a large number of conformations characterize the unfolded state while the folded one is dominated by a single structure, recently a reassessment of the denatured state has been suggested. A growing amount of evidence indicates that not only the folded but also the unfolded state is at least partially organized. Here, we try to answer the question of how different protein dynamics is in folded and unfolded states by performing all-atom molecular dynamics simulations on the model protein Trp-cage. Random matrix theory inspired analysis of the correlation matrices has been carried out. The spectra of these correlation matrices show that the low rank modes of Trp-cage dynamics are outside of the limit expected for a random system both in folded and in unfolded conditions. These findings shed light on the nature of the unfolded state of the proteins, suggesting that it is much less random than previously thought.

Amyloid beta(1-42) in aqueous environments: effects of ionic strength and E22Q (Dutch) mutation.

Development of extracellular plaques characteristic of Alzheimer's disease is related to aggregation of amyloid peptides. The Aß-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, Aß-42 conformational transitions in water have been largely investigated by molecular dynamics. Here we report an all-atom molecular dynamics analysis of the Aß-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 Aß-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 Aß-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 Aß-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.

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.

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.

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon.

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10-N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.

Structure and expression of the atp operon coding for F1F0-ATP synthase from the antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727.

Nonomuraea sp. ATCC 39727 is a poorly characterized actinomycete, producer of the glycopeptide antibiotic A40926. In this study, the nucleotide sequence of the atp operon coding for F1F0-ATP synthase of Nonomuraea sp. ATCC 39727 was determined. It consisted of ten open reading frames arranged in the order atpI (encoding the i protein), orfX, atpB (a subunit), atpE (c subunit), atpF (b subunit), atpH (delta subunit), atpA (alpha subunit), atpG (gamma subunit), atpD (beta subunit) and atpC (epsilon subunit). The orfX coded for a putative small hydrophobic 71 amino acid peptide of unknown function related to several bacterial permeases. Its presence appeared to be a distinctive feature of the atp operon of phylogenetically distant actinobacteria. Transcription of the atp operon was evaluated. The results of northern blot and RT-PCR experiments demonstrated that the atp genes were co-transcribed into a single polycistronic mRNA. Real-time RT-PCR data provided evidence showing that transcription of the atp operon was biphasic during Nonomuraea growth. The amount of the atpD transcript decreased at the end of the exponential growth phase, and then moderately increased during the early stationary phase when, in contrast, the levels of ctaC, encoding the cytochrome c oxidase subunit II, progressively decreased. Western blot analysis confirmed that ATP synthase was also present in the membrane during the stationary phase. These results together with previous data demonstrate that oligomycin-sensitive ATP-driven proton pumping activity remained constant in the stationary phase; in contrast, the activity and cytochrome content of the respiratory enzymes became negligible.

Protonmotive cooperativity in cytochrome c oxidase.

Cooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins. Analysis of proton/electron coupling at the four redox centers, i.e. Cu(A), heme a, heme a(3) and Cu(B), in the purified bovine cytochrome c oxidase in the unliganded, CO-liganded and CN-liganded states is presented. These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers. Heme a (and Cu(A)) exhibits a cooperative proton/electron linkage (Bohr effect). Bohr effect seems also to be associated with the oxygen-reduction chemistry at the heme a(3)-Cu(B) binuclear center. Data on electron transfer in cytochrome c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from Cu(A) to the binuclear center in addition to electron transfer via heme a. A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme a in the proton pump of cytochrome c oxidase is presented. On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined.